In vivo production of cyclic peptides

ABSTRACT

The present invention relates to methods and compositions utilizing inteins to generated libraries of cyclic peptides in vivo.

This application claims the benefit of U.S. Ser. No. 60/187,130, filedMar. 6, 2000.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for generatingintracellular cyclic peptide and protein libraries.

BACKGROUND OF THE INVENTION

Combinatorial libraries of synthetic and natural products are importantsources of molecular information for the development of pharmacologicagents. Linear peptide libraries, containing known and random peptidesequences, are particularly good sources of new and novel compounds fordrug development because of the diversity of structures which can begenerated. Drawbacks to linear peptide libraries are: (1) linearpeptides are generally flexible molecules with entropic limitations onachieving productive biologically active conformations; (2) linearpeptides are susceptible to proteolytic enzymes; and, (3) linearpeptides are inherently instable. For this reason, approaches utilizingconformational and topographical constraints to restrict the number ofconformational states a peptide molecule may assume have been sought.See, for example, Hruby, (1982) Life Sci., 31:189; Hruby, et al., (1990)Biochem. J. 268:249.

Head-to-tail (backbone) peptide cyclization has been used to rigidifystructure and improve in vivo stability of small bioactive peptides (seeCamarero and Muir, (1999) J. Am. Chem. Soc., 121:5597-5598). Animportant consequence of peptide cyclization is retention of biologicalactivity and/or the identification of new classes of pharmacologicalagents. Cyclic peptides have been reported that inhibit T-cell adhesion(Jois, et al. (1999) J. Pept. Res., 53:18-29), PDGF action (Brennand, etal. (1997) FEBS Lett., 413:70-74), and function as new classes of drugs(Kimura et al., (1997) J. Antibiot., 50:373-378; Eriksson, et al.,(1989) Exp. Cell Res., 185:86-100).

Strategies for the preparation of circular polypeptides from linearprecursors have been described. For example, a chemical cross-linkingapproach was used to prepare a backbone cyclized version of bovinepancreatic trypsin inhibitor (Goldenburg and Creighton (1983) J. Mol.Biol., 165:407-413). Other approaches include chemical (Camarero, etal., (1998) Angew. Chem. Int. Ed., 37:347-349; Tam and Lu (1998) Prot.Sci., 7:1583-1592; Camarero and Muir (1997) Chem. Commun.,1997:1369-1370; and Zhang and Tam (1997) J. Am. Chem. Soc.119:2363-2370) and enzymatic (Jackson et al., (1995) J. Am. Chem. Soc.,117:819-820) intramolecular ligation methods which allow linearsynthetic peptides to be efficiently cyclized under aqueous conditions.However, the requirement for synthetic peptide precursors has limitedthese chemical/enzymatic cyclization approaches to systems that are bothex vivo and limited to relatively small peptides.

One solution to this problem has been to generate circular recombinantpeptides and proteins using a native chemical ligation approach. Thisapproach utilizes inteins (internal proteins) to catalyze head-to-tailpeptide and protein ligation in vivo (see, for example, Evans, et al.(1999) J. Biol. Chem. 274:18359-18363; Iwai and Plückthun (1999) FEBSLett. 459:166-172; Wood, et al. (1999) Nature Biotechnology 17:889-892;Camarero and Muir (1999) J. Am. Chem. Soc., 121:5597-5598; and Scott, etal. (1999) Proc. Natl. Acad. Sci. USA, 96:13638-13643).

Inteins are self-splicing proteins that occur as in-frame insertions inspecific host proteins. In a self-splicing reaction, inteins excisethemselves from a precursor protein, while the flanking regions, theexteins, become joined to restore host gene function. Inteins can alsocatalyze a trans-ligation self-splicing reaction. Approaches making useof the trans ligation reaction include splitting the intein into twoparts and reassembling the two parts in vitro, each fused to a differentextein (Southworth, et al., (1998) EMBO J. 17:918-926). A somewhatdifferent approach uses an intein domain, and the reaction is thentriggered with a thiolate nucleophile, such as DTT (Xu, et al., (1998)Protein Sci., 7:2256-2264).

The ability to construct intein fusions to proteins of interest hasfound several applications. For example, inteins can be used inconjunction with an affinity group to purify a desired protein (Wood, etal. (1999) Nature Biotechnology, 17:889-892). Circular recombinantfusion proteins have been created by cloning into a commerciallyavailable intein expression system (Camarero and Muir, (1999) J. Am.Chem. Soc., 121:5597-5598; Iwai and Plückthun (1999) FEBS Lett.459:166-172; and Evans, et al. (1999) J. Biol. Chem. 274:18359-18363).In another approach, a mechanism for in vivo split intein-mediatedcircular ligation of peptides and proteins via permutation of the orderof elements in the fusion protein precursor has been used to expresscyclic products in bacteria (Scott, et al., (1999) Proc. Natl. Acad.Sci. USA, 96:13638-13643).

Cyclic peptide libraries have been generated in phage (Koivunen, et al.,(1995) Biotechnology 13:265-70) and by using the backbone cyclicproteinomimetic approach (Friedler, et al., (1998) Biochemistry,37:5616-22). Methods for modifying inteins for the purpose of creatingcyclic peptides and/or proteins have been recently described (Benkovic,et al., WO 00/36093). It is an object of this invention to utilizeintein function, derived from wild-type or mutant intein structures, togenerate cyclic peptide libraries in vivo. The utilization of mutantintein structures for this purpose are of particular focus since thesehave been optimized for function in the specific context of an inteinscaffold engineered to result in peptide/protein cyclization. Methodsare described for generating, identifying, and utilizing mutants withaltered splicing/cyclization activity for use with cyclicpeptide/protein libraries. Intein-generated cyclic libraries aredescribed for the identification of cyclic peptides/proteins capable ofaltering a given cellular phenotype. Accordingly, it is an object of theinvention to provide compositions and methods useful in the generationof random fusion polypeptide libraries in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts head to tail protein cyclization byreconfigured/engineered intein.

FIG. 1B depicts the mechanism of cyclization by reconfigured/engineeredintein.

FIG. 2A depicts intein catalyzed ligation by the Mxe GyrA intein. In itsnormal configuration, intein catalyzed ligation joins the exteinresidues located at the junction points with each of the two inteinmotifs.

FIG. 2B depicts the outcome of a motif reorganization resulting in theproduction of a cyclic peptide. Motif reorganziation involves providingintein B with its own translational start-codon and placing intein Bamino-terminal to intein A.

FIG. 3A depicts the amino acid sequence of intein Ssp DnaB fromSynechocystis spp. strain PCC6803.

FIG. 3B depicts the amino acid sequence of intein Mxe GyrA fromMycobacterium xenopi.

FIG. 3C depicts the amino acid sequence of intein Ceu CIpP fromChlamydomonas eugametos.

FIG. 3D depicts the amino acid sequence of intein CIV RIR1 from Chiloiridescent virus.

FIG. 3E depicts the amino acid sequence of intein Ctr VMA from Candidatropicalis.

FIG. 3F depicts the amino acid sequence of intein Gth DnaB fromGuillardia theta.

FIG. 3G depicts the amino acid sequence of intein Ppu DnaB from Porphyrapurpurea.

FIG. 3H depicts the amino acid sequence of intein Sce VMA fromSaccharomyces cerevisiae.

FIG. 3I depicts the amino acid sequence of intein Mf1 RecA fromMycobacterium flavescens.

FIG. 3J depicts the amino acid sequence of intein Ssp DnaE fromSynechocystis spp. strain PCC6803.

FIG. 3K depicts the amino acid sequence of intein Mle DnaB fromMycobacterium leprae.

FIG. 3L depicts the amino acid sequence of intein Mja KIbA fromMethanococcus jannaschii.

FIG. 3M depicts the amino acid sequence of intein Pfu KIbA fromPyrococcus furiosus.

FIG. 3N depicts the amino acid sequence of intein Mth RIR1 fromMethanobacterium thermoautotrophicum (delta H strain).

FIG. 3O depicts the amino acid sequence of intein Pfu RIR1-1 fromPyrococcus furiosus.

FIG. 3P depicts the amino acid sequence of intein Psp-GBD Pol fromPyrococcus spp. GB-D.

FIG. 3Q depicts the amino acid sequence of intein Thy Pol-2 fromThermococcus hydrothermalis.

FIG. 3R depicts the amino acid sequence of intein Pfu IF2 fromPyrococcus furiosus.

FIG. 3S depicts the amino acid sequence of intein Pho Lon fromPyrococcus horikoshii OT3.

FIG. 3T depicts the amino acid sequence of intein Mja r-Gyr fromMethanococcus jannaschii.

FIG. 3U depicts the amino acid sequence of intein Pho RFC fromPyrococcus horikoshii OT3.

FIG. 3V depicts the amino acid sequence of intein Pab RFC-2 fromPyrococcus abyssi.

FIG. 3W depicts the amino acid sequence of intein Mja RtcB (Mja Hyp-2)from Methanococcus jannaschii.

FIG. 3X depicts the amino acid sequence of intein Pho VMA fromPyrococcus horikoshii OT3.

FIG. 4A depicts the amino acid sequence of a modified wild-type Ssp DnaBIntein. The DNA sequence is provided in FIG. 4B.

FIGS. 5A and B depict the nucleotide and amino acid sequence of theintein Ssp DnaB J3 template used to generate intein mutants L7-J3,E6-J3, E9-J3, C11-J3 and B8-J3, with improved splicing efficiency. TheJ3 template carries a mutation which results in a amino acid change D toN at position 320. Thus, all mutants based on the J3 template are doublemutants.

FIGS. 5C and D depict the nucleotide and amino acid sequence of inteinmutant L7-J3. L7 has two mutations which result in amino acidchanges: 1) D to N at position 320 and 2) R to K at position 389.

FIGS. 5E and F depict the nucleotide and amino acid sequence of inteinmutant E6-J3. E6 has two mutations which result in amino acidchanges: 1) D to N at position 320 and 2) 1 to V at position 34.

FIGS. 5G and H depict the nucleotide and amino acid sequence of inteinmutant E9-J3. E9 has two mutations which result in amino acidchanges: 1) D to N at position 320 and 2) T to A at position 36.

FIGS. 5I and J depict the nucleotide and amino acid sequence of inteinmutant C11-J3. C11 has two mutations which result in amino acidchanges: 1) D to N at position 320 and 2) S to P at position 23.

FIGS. 5K and L depict the nucleotide and amino acid sequence of inteinmutant B8-J3. B8 has two mutations which result in amino acidchanges: 1) D to N at position 320 and 2) K to R at position 369.

FIGS. 5M and N depict the nucleotide and amino acid sequence of inteinmutant L7-wt, which was generated from an Ssp DnaB wild-type (wt)template. Mutants generated from the wt template carry a single mutationwhich effects splicing efficiency. L7-wt carries a single mutation whichresults in the amino acid change R to K at position 389.

FIGS. 5O and P depict the nucleotide and amino acid sequence of inteinmutant C11-wt. C11-wt has a single mutation which result in the aminoacid change S to P at position 23.

FIGS. 5Q and R depict the nucleotide and amino acid sequence of inteinmutant E6-wt. E6-wt has a single mutation which result in the amino acidchange I to V at position 34.

FIG. 6 depicts the DNA sequence for a N-terminally fused GFP version ofthe Ssp DnaB intein.

FIG. 7 depicts reporter proteins which can be used for the selectionand/or detection of intein-based libraries.

FIG. 8 depicts localization sequences which can be used to target cyclicpeptide libraries.

FIG. 9 depicts a random mutagenesis approach used in the optimization ofintein cyclization function.

FIG. 10 depicts a biotinylation approach for use in a yeast two hybridsystem.

FIG. 11 depicts a single chain antibody approach for use in a yeast twohybrid system.

FIG. 12 depicts the fluorescent reporter system used to quantify inteincyclization. FIG. 12 A depicts GFP split at the loop 3 junction andreversal of the translation order of the N- and C-terminal fragments.The termini are fused using a glycine-serine linker. The GFP ispositioned within the Ssp DnaB intein cyclizationscaffold. Cyclizedproduct reconstitutes both structure and fluorescence of GFP. Inaddition, splicing one-half of the myc epitope onto either side of theloop 3 junction allows for reconstruction of the myc epitope uponcyclization.

FIG. 12B provides the amino acid sequence of DNAB intein cyclizationscaffold with GFP.

FIG. 12C depicts the mechanism of intein catalyzed cyclization ofinverted loop 3 of GFP.

FIG. 12D shows the results from a FACS analysis of the cyclizationefficiency of wild-type Ssp DnaB intein in mammalian cells.

FIG. 12E shows the results from a Western analysis of a Ssp DnaBcatalyzed cyclization in mammalian cells.

FIG. 12F shows the results form a native gel and the contribution to GFPfluorescence. The majority for the fluorescence arises from theformation of cyclized GFP product, bands C and D.

FIG. 13 illustrates a functional screen for isolating randomly-generatedmutants with altered cyclization activity. FIG. 13A depicts a functionalscreen for intein mutants with altered cyclization activity. FIG. 13Bdepicts mutations modeled on the Mxe GyrA intein structure. FIG. 13Cdepicts the sequence alignment of Mxe GyrA and Ssp DnaB inteins. Mutantsare identified in shaded color.

FIG. 13D shows the results from a western analysis of isolated mutants.DnaB mutants E9-J3, E6-J3, C11-J3, L7-J3, and B8-J3 have cyclizationefficiencies were are greater than the J3 starting intein template.

FIG. 14 depicts intein-mediated excision/ligation in mammalian cells.FIG. 14 A depicts constructs in which Ssp DnaB intein is inserted intoloop 3 of GFP (i.e., GAB) or GFP with a C-terminal myc epitope. FIG. 14Bdepicts constructs similar to those shown in 14A, except that the mycepitope half-sites are positioned onto the extreme ends of each splicejunction. FIG. 14C depicts Western blot analysis of lysates fromtransfected Phoenix cells. Lanes 3 and 4 demonstrate efficient splicingwith only slight amounts of unspliced product detected.

FIGS. 15A-D depict a method for detecting cyclic peptides in mammaliancells. FIG. 15A depicts an overview of the method in which cyclicpeptides are detected in mammalian cells expressing a GFP fused inteinscaffold with cyclic peptide inserts. FIGS. 15 B and C depict the MSanalysis of mammalian cell lysates expressing the cyclic peptideproducts from RGD7 (15B) and RGD9 (15C). FIG. 15D depicts an example ofLC/MS fragmentation fingerprinting of the cyclic peptide product of anintein construct.

FIG. 16 depicts the low energy conformers associated with cyclic peptideSRGDGWS.

FIG. 17 depicts the low energy conformers associated with cyclic peptideSRGPGWS.

DETAILED DESCRIPTION OF THE INVENTION

Peptide libraries are an important source of new and novel drugs.However, a number of hurdles must be overcome in order to express andsubsequently screen functional peptides and proteins in cells. Foremostamongst these hurdles is the need to retain biological activity of thepeptides in a cellular environment. To overcome this problem, thepresent invention is directed to fusions of intein motifs and randompeptides such that circular peptides are formed which retain biologicalactivity.

Thus, generally, the present invention provides methods for generatinglibraries of cyclic peptides using inteins. Inteins are self-splicingproteins that occur as in-frame insertions in specific host proteins. Ina self-splicing reaction, inteins excise themselves from a precursorprotein, while the flanking regions, the exteins, become joined via anew peptide bond to form a linear protein. By changing the N to Cterminal orientation of the intein segments, the ends of the exteinjoin, forming a cyclized extein. FIG. 1 illustrates intein catalyzedjoining of extein residues located at the junction points with each ofthe two intein motifs.

Because intein function is not strongly influenced by the nature of theextein polypeptide sequences located between them, standard recombinantmethods can be used to insert random libraries into this position.Placement of these intein libraries into any number of delivery systemsallows for the subsequent expression of unique cyclic peptides withinindividual cells. Such cells can then be screened to identify peptidesof interest.

Accordingly, the present invention provides fusion polypeptidescomprising intein motifs and peptides.

By “fusion polypeptide” or “fusion peptide” or grammatical equivalentsherein is meant a protein composed of a plurality of protein components,that while typically unjoined in their native state, are joined by theirrespective amino and carboxyl termini through a peptide linkage to forma single continuous polypeptide. “Protein” in this context includesproteins, polypeptides and peptides. Plurality in this context means atleast two, and preferred embodiments generally utilize two components.It will be appreciated that the protein components can be joineddirectly or joined through a peptide linker/spacer as outlined below. Inaddition, as outlined below, additional components such as fusionpartners including targeting sequences, etc. may be used.

The present invention provides fusion proteins of intein motifs andrandom peptides. By “inteins”, or “intein motifs”, or “intein domains”,or grammatical equivalents herein is meant a protein sequence which,during protein splicing, is excised from a protein precursor. Alsoincluded within in the definition of intein motifs are DNA sequencesencoding inteins and mini-inteins.

Many inteins, are bifunctional proteins mediating both protein splicingand DNA cleavage. Such elements consist of a protein splicing domaininterrupted by an endonuclease domain. Because endonuclease activity isnot required for protein splicing, mini-inteins with accurate splicingactivity can be generated by deletion of this central domain (Wood, etal., (1999) Nature Biotechnology, 17:889-892), hereby incorporated byreference.

Protein splicing involves four nucleophilic displacements by threeconserved splice junction residues. These residues, located near theintein/extein junctions, include the initial cysteine, serine, orthreonine of the intein, which intiates splicing with an acyl shift. Theconserved cysteine, serine, or threonine of the extein, which ligatesthe exteins through nucleophilic attack, and the conserved C-terminalhistidine and asparagine of the intein, which releases the intein fromthe ligated exteins through succinimide formation. See Wood, et al.,(1999) supra.

Inteins also catalyze a trans-ligation reaction. The ability of inteinfunction to be reconstituted in trans by spatially separated inteindomains suggests that reorganization of the self-splicing motifs can beused to produce peptides with a circular topology.

In a preferred embodiment, the translational order in which the N- andC-terminal intein motifs are normally synthesized within a polypetidechain is reversed. Generally, a reversal in the translational order inwhich the N- and C-terminal intein motifs are synthesized should notfundamentally change the enzymatic function of the intein. However, thelocation of the intervening peptide's amino and carboxy termini arealtered in such a way that the product of the intein ligation reactionis no longer linear, but rather is cyclized. FIG. 2 illustrates theoutcome of a motif reorganization in which intein B has been given itsown translational start codon and placed amino-terminal to intein A. Toeffectively express unique peptides in cells, fusion polypetidescomprising a C-terminal motif, a peptide and a N-terminal motif areselected or designed for the production of random libraries of cyclicpeptides in vivo.

In a preferred embodiment, the fusion polypeptide is designed with theprimary sequence from the N-terminus comprising I_(A)-target-I_(B).I_(A) is defined herein as the C-terminal intein motif, I_(B) is definedherein as the N-terminal intein motif and target is defined herein as apeptide. DNA sequences encoding the inteins may be obtained from aprokaryotic DNA sequence, such as a bacterial DNA sequence, or aeukaryotic DNA sequence, such as a yeast DNA sequence. The InteinRegistry includes a list of all experimental and theoretical inteinsdiscovered to date and submitted to the registry(http://www.neb.com/inteins/int_reg.html).

In a preferred embodiment, fusion polypeptides are designed using inteinmotifs selected from organisms belonging to the Eucarya and Eubacteria,with the intein Ssp DnaB (GenBank accession number Q55418) beingparticularly preferred. The GenBank accession numbers for other inteinproteins and nucleic acids include, but are not limited to: Ceu CIpP(GenBank accession number P42379); CIV RIR1 (T03053); Ctr VMA (GenBankaccession number A46080); Gth DnaB (GenBank accession number O78411);Ppu DnaB (GenBank accession number P51333); Sce VMA (GenBank accessionnumber PXBYVA); Mf1 RecA (GenBank accession number not given); Mxe GyrA(GenBank accession number P72065); Ssp DnaE (GenBank accession numberS76958 & S75328); and Mle DnaB (GenBank accession number CAA17948.1)

In other embodiments, inteins with alternative splicing mechanisms arepreferred (see Southworth, et al., (2000) EMBO J., 19:5019-26). TheGenBank accession numbers for inteins with alternative splicingmechanisms include, but are not limited to: Mja KIbA (GenBank accessionnumber Q58191); and, Pfu KIbA (PF_(—)949263 in UMBI).

In yet other embodiments, inteins from thermophilic organisms are used.Random mutagenesis or directed evolution (i.e. PCR shuffling, etc.) ofinteins from these organisms could lead to the isolation of temperaturesensitive mutants. Thus, inteins from thermophiles (i.e., Archaea) whichfind use in the invention are: Mth RIR1 (GenBank accession numberG69186); Pfu RIR1-1 (AAB36947.1); Psp-GBD Pol (GenBank accession numberAAA67132.1); Thy Pol-2 (GenBank accession number CAC18555.1); Pfu IF2(PF_(—)1088001 in UMBI); Pho Lon Baa29538.1); Mja r-Gyr (GenBankaccession number G64488); Pho RFC (GenBank accession number F71231); PabRFC-2 (GenBank accession number C75198); Mja RtcB (also referred to asMja Hyp-2; GenBank accession number Q58095); and, Pho VMA (NT01 PH1971in Tigr).

Preferred fusion polypeptides of the invention increase the efficiencyof the cyclization reaction by selecting or designing intein motifs withaltered cyclization activity when expressed in vivo. In a preferredembodiment, the fusion polypeptides of the invention employ the DNAsequence encoding the Synechocystis ssp. strain PCC6803 DnaB intein. Aparticularly preferred fusion polypeptide structure is illustrated inFIGS. 4A and 4B.

In a preferred embodiment, fusion polypeptides are designed using mutantintein sequences with altered cyclization activity as described below.Preferred mutant intein sequences include, but are not limited, to thoseshown in FIG. 5.

In a preferred embodiment, the fusion polypeptides of the inventioncomprise peptides. That is, the fusion polypeptides of the invention aretranslation products of nucleic acids. In this embodiment, nucleic acidsare introduced into cells, and the cells express the nucleic acids toform peptides.] Generally, peptides ranging from about 4 amino acids inlength to about 100 amino acids may be used, with peptides ranging fromabout 5 to about 50 being preferred, with from about 5 to about 30 beingparticularly preferred and from about 6 to about 20 being especiallypreferred.

In a preferred embodiment, the fusion polypeptides of the inventioncomprise random peptides. By “random peptides” herein is meant that eachpeptide consists of essentially random amino acids. Since generallythese random peptides (or nucleic acids, discussed below) are chemicallysynthesized, they may incorporate any amino acid at any position. Thesynthetic process can be designed to generate randomized proteins toallow the formation of all or most of the possible combinations over thelength of the sequence, thus forming a library of randomized peptides.

In a preferred embodiment, the fusion polypeptides of the inventioncomprise peptides derived from a cDNA library.

The fusion polypeptide preferably includes additional components,including, but not limited to, reporter proteins and fusion partners.

In a preferred embodiment, the fusion polypeptides of the inventioncomprise a reporter protein. By “reporter protein” or grammaticalequivalents herein is meant a protein that by its presence in or on acell or when secreted in the media allow the cell to be distinguishedfrom a cell that does not contain the reporter protein. As describedherein, the cell usually comprises a reporter gene that encodes thereporter protein.

Reporter genes fall into several classes, as outlined above, including,but not limited to, detection genes, indirectly detectable genes, andsurvival genes. See FIG. 6.

In a preferred embodiment, the reporter protein is a detectable protein.A “detectable protein” or “detection protein” (encoded by a detectableor detection gene) is a protein that can be used as a direct label; thatis, the protein is detectable (and preferably, a cell comprising thedetectable protein is detectable) without further manipulations orconstructs. As outlined herein, preferred embodiments of screeningutilize cell sorting (for example via FACS) to detect reporter (and thuspeptide library) expression. Thus, in this embodiment, the proteinproduct of the reporter gene itself can serve to distinguish cells thatare expressing the detectable gene. In this embodiment, suitabledetectable genes include those encoding autofluorescent proteins.

Detectable enzyme products resulting from the intein cyclizationreaction may also be used to detect cells that are expressing thedetectable product. Examples of enzymes which can be used includeluciferase, β-galactosidase, β-lactamase, puromycin resistance protein,etc.

As is known in the art, there are a variety of autofluorescent proteinsknown; these generally are based on the green fluorescent protein (GFP)from Aequorea and variants thereof; including, but not limited to, GFP,(Chalfie, et al., “Green Fluorescent Protein as a Marker for GeneExpression,” Science 263(5148):802-805 (1994)); enhanced GFP (EGFP;Clontech-Genbank Accession Number U55762)), blue fluorescent protein(BFP; Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8thFloor, Montreal (Quebec) Canada H3H 1J9; Stauber, R. H. Biotechniques24(3):462-471 (1998); Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182(1996)), enhanced yellow fluorescent protein (EYFP; ClontechLaboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303)and red fluorescent protein. In addition, there are recent reports ofautofluorescent proteins from Renilla and Ptilosarcus species. See WO92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; U.S. Pat.No. 5,292,658; U.S. Pat. No. 5,418,155; U.S. Pat. No. 5,683,888; U.S.Pat. No. 5,741,668; U.S. Pat. No. 5,777,079; U.S. Pat. No. 5,804,387;U.S. Pat. No. 5,874,304; U.S. Pat. No. 5,876,995; and U.S. Pat. No.5,925,558; all of which are expressly incorporated herein by reference.

Preferred fluorescent molecules include but are not limited to greenfluorescent protein (GFP; from Aquorea and Renilla species), bluefluorescent protein (BFP), yellow fluorescent protein (YFP), and redfluorescent protein (RFP).

In a preferred embodiment, the reporter protein is Aequorea greenfluorescent protein or one of its variants; see Cody et al.,Biochemistry 32:1212-1218 (1993); and Inouye and Tsuji, FEBS Lett.341:277-280 (1994), both of which are expressly incorporated byreference herein. However, as is understood by those in the art,fluorescent proteins from other species may be used.

Accordingly, the present invention provides fusions of green fluorescentprotein (GFP) and random peptides. By “green fluorescent protein” or“GFP” herein is meant a protein with at least 30% sequence identity toGFP and exhibits fluorescence at 490 to 600 nm. The wild-type GFP is 238amino acids in length, contains a modified tripeptide fluorophore buriedinside a relatively rigid β-can structure which protects the fluorophorefrom the solvent, and thus solvent quenching. See Prasher et al., Gene111(2):229-233 (1992); Cody et al., Biochem. 32(5):1212-1218 (1993);Ormo et al, Science 273:1392-1395 (1996); and Yang et al., Nat. Biotech.14:1246-1251 (1996), all of which are hereby incorporated by referencein their entirety). Included within the definition of GFP arederivatives of GFP, including amino acid substitutions, insertions anddeletions. See for example WO 98/06737 and U.S. Pat. No. 5,777,079, bothof which are hereby incorporated by reference in their entirety.Accordingly, the GFP proteins utilized in the present invention may beshorter or longer than the wild type sequence. Thus, in a preferredembodiment, included within the definition of GFP proteins are portionsor fragments of the wild type sequence. For example, GFP deletionmutants can be made. At the N-terminus, it is known that only the firstamino acid of the protein may be deleted without loss of fluorescence.At the C-terminus, up to 7 residues can be deleted without loss offluorescence; see Phillips et al., Current Opin. Structural Biol. 7:821(1997)).

For fusions involving fluorescent proteins other than GFP, proteins withat least 24% sequence homology to YFP, RFP, BFP are included with thescope of the present invention.

In a preferred embodiment, intein A is fused to the N-terminus of GFP.The fusion can be direct, i.e. with no additional residues between theC-terminus of intein A and the N-terminus of GFP, or indirect; that is,intervening amino acids are inserted between the N-terminus of GFP andthe C-terminus of intein A. See FIG. 7.

In a preferred embodiment, intein B is fused to the C-terminus of GFP.As above for N-terminal fusions, the fusion can be direct or indirect.

In a preferred embodiment, the reporter protein is an indirectlydetectable protein. As for the reporter proteins, cells that contain theindirectly detectable protein can be distinguished from those that donot; however, this is as a result of a secondary event. For example, apreferred embodiment utilizes “enzymatically detectable” reporters thatcomprise enzymes that will act on chromogenic, and particularlyfluorogenic, substrates, to generate fluorescence, such as luciferase,β-galactosidase, and β-lactamase. Alternatively, the indirectlydetectable protein may require a recombinant construct in a cell thatmay be activated by the reporter; for example, transcription factors orinducers that will bind to a promoter linked to an autofluorescentprotein such that transcription of the autofluorescent protein occurs.

In a preferred embodiment, the indirectly detectable protein is aDNA-binding protein which can bind to a DNA binding site and activatetranscription of an operably linked reporter gene. The reporter gene canbe any of the detectable genes, such as green fluorescent protein, orany of the survival genes, outlined herein. The DNA binding site(s) towhich the DNA binding protein is binding is (are) placed proximal to abasal promoter that contains sequences required for recognition by thebasic transcription machinery (e.g., RNA polymerase II). The promotercontrols expression of a reporter gene. Following introduction of thischimeric reporter construct into an appropriate cell, an increase of thereporter gene product provides an indication that the DNA bindingprotein bound to its DNA binding site and activated transcription.Preferably, in the absence of the DNA binding protein, no reporter geneproduct is made. Alternatively, a low basal level of reporter geneproduct may be tolerated in the case when a strong increase in reportergene product is observed upon the addition of the DNA binding protein,or the DNA binding protein encoding gene. It is well known in the art togenerate vectors comprising DNA binding site(s) for a DNA bindingprotein to be analyzed, promoter sequences and reporter genes.

In a preferred embodiment, the DNA-binding protein is a cell typespecific DNA binding protein which can bind to a nucleic acid bindingsite within a promoter region to which endogenous proteins do not bindat all or bind very weakly. These cell type specific DNA-bindingproteins comprise transcriptional activators, such as October-2 [Muelleret al., Nature 336(6199):544-51 (1988)] which e.g., is expressed inlymphoid cells and not in fibroblast cells. Expression of this DNAbinding protein in HeLa cells, which usually do not express thisprotein, is sufficient for a strong transcriptional activation of B-cellspecific promoters, comprising a DNA binding site for October-2 (Muelleret al., supra).

In a preferred embodiment, the indirectly detectable protein is aDNA-binding/transcription activator fusion protein which can bind to aDNA binding site and activate transcription of an operably linkedreporter gene. Briefly, transcription can be activated through the useof two functional domains of a transcription activator protein; a domainor sequence of amino acids that recognizes and binds to a nucleic acidsequence, i.e. a nucleic acid binding domain, and a domain or sequenceof amino acids that will activate transcription when brought intoproximity to the target sequence. Thus the transcriptional activationdomain is thought to function by contacting other proteins required intranscription, essentially bringing in the machinery of transcription.It must be localized at the target gene by the nucleic acid bindingdomain, which putatively functions by positioning the transcriptionalactivation domain at the transcriptional complex of the target gene.

The DNA binding domain and the transcriptional activator domain can beeither from the same transcriptional activator protein, or can be fromdifferent proteins (see McKnight et al., Proc. Natl. Acad. Sci. USA89:7061 (1987); Ghosh et al., J. Mol. Biol. 234(3):610-619 (1993); andCurran et al., 55:395 (1988)); A variety of transcriptional activatorproteins comprising an activation domain and a DNA binding domain areknown in the art.

In a preferred embodiment the DNA-binding/transcription activator fusionprotein is a tetracycline repressor protein (TetR)-VP16 fusion protein.This bipartite fusion protein consists of a DNA binding domain (TetR)and a transcription activation domain (VP16). TetR binds with highspecificity to the tetracycline operator sequence, (tetO). The VP16domain is capable of activating gene expression of a gene of interest,provided that it is recruited to a functional promoter. Employing atetracycline repressor protein (TetR)-VP16 fusion protein, a suitableeukaryotic expression system which can be tightly controlled by theaddition or omission of tetracycline or doxycycline has been described(Gossen and Bujard, Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551; Gossenet al., Science 268:1766-1769 (1995)].

It is an object of the instant application to fuse intein amino acidsequences to DNA-binding/transcription activator proteins and/or toDNA-binding/transcription activator fusion proteins. N-terminal andC-terminal fusions are all contemplated. The site of fusion may bedetermined based on the structure of DNA-binding/transcription activatorfusion protein, which are determined [e.g., TetR; see Orth et al., J.Mol. Biol. 285(2):455-61 (1999); Orth et al., J. Mol. Biol.279(2):439-47 (1998); Hinrichs et al., Science 264(5157):418-20 (1994);and Kisker et al., J. Mol. Biol. 247(2):260-80 (1995)].

In a preferred embodiment, the reporter protein is a survival protein.By “survival protein”, “selection protein” or grammatical equivalentsherein is meant a protein without which the cell cannot survive, such asdrug resistance genes. As described herein, the cell usually does notnaturally contain an active form of the survival protein which is usedas a scaffold protein. As further described herein, the cell usuallycomprises a survival gene that encodes the survival protein.

The expression of a survival protein is usually not quantified in termsof protein activity, but rather recognized by conferring acharacteristic phenotype onto a cell which comprises the respectivesurvival gene or selection gene. Such survival genes may provideresistance to a selection agent (i.e., an antibiotic) to preferentiallyselect only those cells which contain and express the respectivesurvival gene. The variety of survival genes is quite broad andcontinues to grow (for review see Kriegler, Gene Transfer andExpression: A Laboratory Manual, W.H. Freeman and Company, New York,1990). Typically, the DNA containing the resistance-conferring phenotypeis transfected into a cell and subsequently the cell is treated withmedia containing the concentration of drug appropriate for the selectivesurvival and expansion of the transfected and now drug-resistant cells.

Selection agents such as ampicillin, kanamycin and tetracycline havebeen widely used for selection procedures in prokaryotes [e.g., seeWaxman and Strominger, Annu. Rev. Biochem. 52:825-69 (1983); Davies andSmith, Annu. Rev. Microbiol. 32:469-518 (1978); and Franklin, BiochemJ., 105(1):371-8 (1967)]. Suitable selection agents for the selection ofeukaryotic cells include, but are not limited to, blasticidin [Izumi etal., Exp. Cell Res., 197(2):229-33 (1991); Kimura et al., Biochim.Biophys. Acta 1219(3):653-9 (1994); Kimura et al., Mol. Gen. Genet.242(2):121-9 (1994)], histidinol D [Hartman and Mulligan; Proc. Natl.Acad. Sci. U.S.A., 85(21):8047-51 (1988)], hygromycin [Gritz and Davies,Gene 25(2-3):179-88 (1983); Sorensen et al., Gene 112(2):257-60 (1992)],neomycin [Davies and Jimenez, Am. J. Trop. Med. Hyg., 29(5Suppl):1089-92 (1980); Southern and Berg, J. Mol. Appl. Genet.,1(4):327-41 (19820], puromycin [de la Luna et al., Gene 62(1):121-6(1988)] and bleomycin/phleomycin/zeocin antibiotics [Mulsant et al.,Somat Cell. Mol. Genet. 14(3):243-52 (1988).

Survival genes encoding enzymes mediating such a drug-resistantphenotype and protocols for their use are known in the art (seeKriegler, supra). Suitable survival genes include, but are not limitedto thymidine kinase [TK; Wigler et al., Cell 11:233 (1977)], adeninephosphoribosyltransferase [APRT; Lowry et al., Cell 22:817 (1980);Murray et al., Gene 31:233 (1984); Stambrook et al., Som. Cell. Mol.Genet. 4:359 (1982)], hypoxanthine-guanine phosphoribosyltransferase[HGPRT; Jolly et al., Proc. Natl. Acad. Sci. U.S.A. 80:477 (1983)],dihydrofolate reductase [DHFR; Subramani et al., Mol. Cell. Biol. 1:854(1985); Kaufman and Sharp, J. Mol. Biol. 159:601 (1982); Simonsen andLevinson, Proc. Natl. Acad. Sci. U.S.A. 80:2495 (1983)] aspartatetranscarbamylase [Ruiz and Wahl, Mol. Cell. Biol. 6:3050 (1986)],ornithine decarboxylase [Chiang and McConlogue, Mol. Cell. Biol. 8:764(1988)], aminoglycoside phosphotransferase [Southern and Berg, Mol.Appl. Gen. 1:327 (1982); Davies and Jiminez, supra],hygromycin-B-phosphotransferase [Gritz and Davies, supra; Sugden et al.,Mol. Cell. Biol. 5:410 (1985); Palmer et al., Proc. Natl. Acad. Sci.U.S.A. 84:1055 (1987)], xanthine-guanine phosphoribosyltransferase[Mulligan and Berg, Proc. Natl. Acad. Sci. U.S.A. 78:2072 (1981)],tryptophan synthetase [Hartman and Mulligan, Proc. Natl. Acad. Sci.U.S.A. 85:8047 (1988)], histidinol dehydrogenase (Hartman and Mulligan,supra), multiple drug resistance biochemical marker [Kane et al., Mol.Cell. Biol. 8:3316 (1988); Choi et al., Cell 53:519 (1988)], blasticidinS deaminase [Izumi et al., Exp. Cell. Res. 197(2):229-33 (1991)],bleomycin hydrolase [Mulsant et al., supra], andpuromycin-N-acetyl-transferase [Lacalle et al., Gene 79(2):375-80(1989)],

In another preferred embodiment, the survival protein is blasticidin Sdeaminase, which is encoded by the bsr gene [Izumi et al., Exp. Cell.Res. 197(2):229-33 (1991)]. When transferred into almost any cell, thisdominant selectable gene confers resistance to media comprising theantibiotic blasticidin S. Blasticidin S deaminase encoding genes havebeen cloned. They are used widely as a selectable marker on variousvectors and the nucleotide sequences are available (e.g., see GenBankaccession numbers D83710, U75992, and U75991).

It is an object of the instant application to fuse intein motifsequences to blasticidin S deaminase. N-terminal and C-terminal fusionsare all contemplated. The site of fusion may be determined based on thestructure of Aspergillus terreus blasticidin S deaminase, which has beendetermined (Nakasako et al., Acta Crystallogr. D. Biol. Crystallogr.55(Pt2):547-8 (1999)]. Also, internal fusions can be done; see PCTUS99/23715, hereby incorporated by reference.

In another preferred embodiment, the survival protein ispuromycin-N-acetyl-transferase, which is encoded by the pac gene[Lacalle et al., Gene 79(2):375-80 (1989)]. When transferred into almostany cell, this dominant selectable gene confers resistance to mediacomprising puromycin. A puromycin-N-acetyltransferase encoding gene hasbeen cloned. It is used widely as a selectable marker on various vectorsand the nucleotide sequences are available (e.g., see GenBank accessionnumbers Z75185 and M25346).

It is an object of the instant application to fuse intein motifsequences puromycin-N-acetyl-transferase. N-terminal and C-terminal,dual N- and C-terminal and one or more internal fusions are allcontemplated.

In a preferred embodiment, in addition to the intein motifs andpeptides, the fusion polypeptides of the present invention preferablyinclude additional components, including, but not limited to, fusionpartners.

By “fusion partner” herein is meant a sequence that is associated withthe fusion polypeptide that confers upon all members of the library inthat class a common function or ability. Fusion partners can beheterologous (i.e. not native to the host cell), or synthetic (notnative to any cell). Suitable fusion partners include, but are notlimited to: a) targeting sequences, defined below, which allow thelocalization of the peptide into a subcellular or extracellularcompartment; b) rescue sequences as defined below, which allow thepurification or isolation of either the peptides or the nucleic acidsencoding them; or c), any combination of a) and b).

In a preferred embodiment, the fusion partner is a targeting sequence.As will be appreciated by those in the art, the localization of proteinswithin a cell is a simple method for increasing effective concentrationand determining function. For example, RAF1 when localized to themitochondrial membrane can inhibit the anti-apoptotic effect of BCL-2.Similarly, membrane bound Sos induces Ras mediated signaling inT-lymphocytes. These mechanisms are thought to rely on the principle oflimiting the search space for ligands, that is to say, the localizationof a protein to the plasma membrane limits the search for its ligand tothat limited dimensional space near the membrane as opposed to the threedimensional space of the cytoplasm. Alternatively, the concentration ofa protein can also be simply increased by nature of the localization.Shuttling the proteins into the nucleus confines them to a smaller spacethereby increasing concentration. Finally, the ligand or target maysimply be localized to a specific compartment, and inhibitors must belocalized appropriately.

Thus, suitable targeting sequences include, but are not limited to,binding sequences capable of causing binding of the expression productto a predetermined molecule or class of molecules while retainingbioactivity of the expression product, (for example by using enzymeinhibitor or substrate sequences to target a class of relevant enzymes);sequences signalling selective degradation, of itself or co-boundproteins; and signal sequences capable of constitutively localizing thepeptides to a predetermined cellular locale, including a) subcellularlocations such as the Golgi, endoplasmic reticulum, nucleus, nucleoli,nuclear membrane, mitochondria, chloroplast, secretory vesicles,lysosome, and cellular membrane; and b) extracellular locations via asecretory signal. Particularly preferred is localization to eithersubcellular locations or to the outside of the cell via secretion. SeeFIG. 8.

In a preferred embodiment, the targeting sequence is a nuclearlocalization signal (NLS). NLSs are generally short, positively charged(basic) domains that serve to direct the entire protein in which theyoccur to the cell's nucleus. Numerous NLS amino acid sequences have beenreported including single basic NLS's such as that of the SV40 (monkeyvirus) large T Antigen (Pro Lys Lys Lys Arg Lys Val), Kalderon (1984),et al., Cell, 39:499-509; the human retinoic acid receptor-β nuclearlocalization signal (ARRRRP); NFβB p50 (EEVQRKRQKL; Ghosh et al., Cell62:1019 (1990); NFβB p65 (EEKRKRTYE; Nolan et al., Cell 64:961 (1991);and others (see for example Boulikas, J. Cell. Biochem. 55(1):32-58(1994), hereby incorporated by reference) and double basic NLS'sexemplified by that of the Xenopus (African clawed toad) protein,nucleoplasmin (Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gin AlaLys Lys Lys Lys Leu Asp), Dingwall, et al., Cell, 30:449-458, 1982 andDingwall, et al., J. Cell Biol., 107:641-849; 1988). Numerouslocalization studies have demonstrated that NLSs incorporated insynthetic peptides or grafted onto reporter proteins not normallytargeted to the cell nucleus cause these peptides and reporter proteinsto be concentrated in the nucleus. See, for example, Dingwall, andLaskey, Ann, Rev. Cell Biol., 2:367-390, 1986; Bonnerot, et al., Proc.Natl. Acad. Sci. USA, 84:6795-6799, 1987; Galileo, et al., Proc. Natl.Acad. Sci. USA, 87:458-462, 1990.

In a preferred embodiment, the targeting sequence is a membraneanchoring signal sequence. This is particularly useful since manyparasites and pathogens bind to the membrane, in addition to the factthat many intracellular events originate at the plasma membrane. Thus,membrane-bound peptide libraries are useful for both the identificationof important elements in these processes as well as for the discovery ofeffective inhibitors. The invention provides methods for presenting therandomized expression product extracellularly or in the cytoplasmicspace. For extracellular presentation, a membrane anchoring region isprovided at the carboxyl terminus of the peptide presentation structure.The randomized expression product region is expressed on the cellsurface and presented to the extracellular space, such that it can bindto other surface molecules (affecting their function) or moleculespresent in the extracellular medium. The binding of such molecules couldconfer function on the cells expressing a peptide that binds themolecule. The cytoplasmic region could be neutral or could contain adomain that, when the extracellular randomized expression product regionis bound, confers a function on the cells (activation of a kinase,phosphatase, binding of other cellular components to effect function).Similarly, the randomized expression product-containing region could becontained within a cytoplasmic region, and the transmembrane region andextracellular region remain constant or have a defined function.

Membrane-anchoring sequences are well known in the art and are based onthe genetic geometry of mammalian transmembrane molecules. Peptides areinserted into the membrane based on a signal sequence (designated hereinas ssTM) and require a hydrophobic transmembrane domain (herein TM). Thetransmembrane proteins are inserted into the membrane such that theregions encoded 5′ of the transmembrane domain are extracellular and thesequences 3′ become intracellular. Of course, if these transmembranedomains are placed 5′ of the variable region, they will serve to anchorit as an intracellular domain, which may be desirable in someembodiments. ssTMs and TMs are known for a wide variety of membranebound proteins, and these sequences may be used accordingly, either aspairs from a particular protein or with each component being taken froma different protein, or alternatively, the sequences may be synthetic,and derived entirely from consensus as artificial delivery domains.

As will be appreciated by those in the art, membrane-anchoringsequences, including both ssTM and TM, are known for a wide variety ofproteins and any of these may be used. Particularly preferredmembrane-anchoring sequences include, but are not limited to, thosederived from CD8, ICAM-2, IL-8R, CD4 and LFA-1.

Useful sequences include sequences from: 1) class I integral membraneproteins such as IL-2 receptor β-chain (residues 1-26 are the signalsequence, 241-265 are the transmembrane residues; see Hatakeyama et al.,Science 244:551 (1989) and von Heijne et al, Eur. J. Biochem. 174:671(1988)) and insulin receptor β-chain (residues 1-27 are the signal,957-959 are the transmembrane domain and 960-1382 are the cytoplasmicdomain; see Hatakeyama, supra, and Ebina et al., Cell 40:747 (1985)); 2)class II integral membrane proteins such as neutral endopeptidase(residues 29-51 are the transmembrane domain, 2-28 are the cytoplasmicdomain; see Malfroy et al., Biochem. Biophys. Res. Commun. 144:59(1987)); 3) type III proteins such as human cytochrome P450 NF25(Hatakeyama, supra); and 4) type IV proteins such as humanP-glycoprotein (Hatakeyama, supra). Particularly preferred are CD8 andICAM-2. For example, the signal sequences from CD8 and ICAM-2 lie at theextreme 5′ end of the transcript. These consist of the amino acids 1-32in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP; Nakauchi et al.,PNAS USA 82:5126 (1985) and 1-21 in the case of ICAM-2(MSSFGYRTLTVALFTLICCPG; Staunton et al., Nature (London) 339:61 (1989)).These leader sequences deliver the construct to the membrane while thehydrophobic transmembrane domains, placed 3′ of the random peptideregion, serve to anchor the construct in the membrane. Thesetransmembrane domains are encompassed by amino acids 145-195 from CD8(PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR; Nakauchi, supra)and 224-256 from ICAM-2 (MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR; Staunton,supra).

Alternatively, membrane anchoring sequences include the GPI anchor,which results in a covalent bond between the molecule and the lipidbilayer via a glycosyl-phosphatidylinositol bond for example in DAF(PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT, with the bolded serine the siteof the anchor; see Homans et al., Nature 333(6170):269-72 (1988), andMoran et al., J. Biol. Chem. 266:1250 (1991)). In order to do this, theGPI sequence from Thy-1 can be cassetted 3′ of the variable region inplace of a transmembrane sequence.

Similarly, myristylation sequences can serve as membrane anchoringsequences. It is known that the myristylation of c-src recruits it tothe plasma membrane. This is a simple and effective method of membranelocalization, given that the first 14 amino acids of the protein aresolely responsible for this function: MGSSKSKPKDPSQR (see Cross et al.,Mol. Cell. Biol. 4(9):1834 (1984); Spencer et al., Science 262:1019-1024(1993), both of which are hereby incorporated by reference). This motifhas already been shown to be effective in the localization of reportergenes and can be used to anchor the zeta chain of the TCR. This motif isplaced 5′ of the variable region in order to localize the construct tothe plasma membrane. Other modifications such as palmitoylation can beused to anchor constructs in the plasma membrane; for example,palmitoylation sequences from the G protein-coupled receptor kinase GRK6sequence (LLQRLFSRQDCCGNCSDSEEELPTRL, with the bold cysteines beingpalmitolyated; Stoffel et al., J. Biol. Chem 269:27791 (1994)); fromrhodopsin (KQFRNCMLTSLCCGKNPLGD; Barnstable et al., J. Mol. Neurosci.5(3):207 (1994)); and the p21H-ras 1 protein (LNPPDESGPGCMSCKCVLS; Caponet al., Nature 302:33 (1983)).

In a preferred embodiment, the targeting sequence is a lysozomaltargeting sequence, including, for example, a lysosomal degradationsequence such as Lamp-2 (KFERQ; Dice, Ann. N.Y. Acad. Sci. 674:58(1992); or lysosomal membrane sequences from Lamp-1(MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI, Uthayakumar et al., Cell. Mol.Biol. Res. 41:405 (1995)) or Lamp-2(LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF, Konecki et al., Biochem. Biophys.Res. Comm. 205:1-5 (1994), both of which show the transmembrane domainsin italics and the cytoplasmic targeting signal underlined).

Alternatively, the targeting sequence may be a mitrochondriallocalization sequence, including mitochondrial matrix sequences (e.g.yeast alcohol dehydrogenase III; MLRTSSLFTRRVQPSLFSRNILRLQST; Schatz,Eur. J. Biochem. 165:1-6 (1987)); mitochondrial inner membrane sequences(yeast cytochrome c oxidase subunit IV; MLSLRQSIRFFKPATRTLCSSRYLL;Schatz, supra); mitochondrial intermembrane space sequences (yeastcytochrome c1;MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYADSLTAEAMTA;Schatz, supra) or mitochondrial outer membrane sequences (yeast 70 kDouter membrane protein; MKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQQRGKK;Schatz, supra).

The target sequences may also be endoplasmic reticulum sequences,including the sequences from calreticulin (KDEL; Pelham, Royal SocietyLondon Transactions B; 1-10 (1992)) or adenovirus E3/19K protein(LYLSRRSFIDEKKMP; Jackson et al., EMBO J. 9:3153 (1990).

Furthermore, targeting sequences also include peroxisome sequences (forexample, the peroxisome matrix sequence from Luciferase; SKL; Keller etal., PNAS USA 4:3264 (1987)); farnesylation sequences (for example,P21H-ras 1; LNPPDESGPGCMSCKCVLS, with the bold cysteine farnesylated;Capon, supra); geranylgeranylation sequences (for example, proteinrab-5A; LTEPTQPTRNQCCSN, with the bold cysteines geranylgeranylated;Farnsworth, PNAS USA 91:11963 (1994)); or destruction sequences (cyclinB1; RTALGDIGN; Klotzbucher et al., EMBO J. 1:3053 (1996)).

In a preferred embodiment, the targeting sequence is a secretory signalsequence capable of effecting the secretion of the peptide. There are alarge number of known secretory signal sequences which are placed 5′ tothe variable peptide region, and are cleaved from the peptide region toeffect secretion into the extracellular space. Secretory signalsequences and their transferability to unrelated proteins are wellknown, e.g., Silhavy, et al. (1985) Microbiol. Rev. 49, 398-418. This isparticularly useful to generate a peptide capable of binding to thesurface of, or affecting the physiology of, a target cell that is otherthan the host cell, e.g., the cell infected with the retrovirus. In apreferred approach, a fusion polypeptide is configured to contain, inseries, a secretion signal peptide-intein B motif-randomized librarysequence-intein A. See FIG. 8. In this manner, target cells grown in thevicinity of cells caused to express the library of peptides, are bathedin secreted peptide. Target cells exhibiting a physiological change inresponse to the presence of a peptide, e.g., by the peptide binding to asurface receptor or by being internalized and binding to intracellulartargets, and the secreting cells are localized by any of a variety ofselection schemes and the peptide causing the effect determined.Exemplary effects include variously that of a designer cytokine (i.e., astem cell factor capable of causing hematopoietic stem cells to divideand maintain their totipotential), a factor causing cancer cells toundergo spontaneous apoptosis, a factor that binds to the cell surfaceof target cells and labels them specifically, etc.

Suitable secretory sequences are known, including signals from IL-2(MYRMQLLSCIALSLALVTNS; Villinger et al., J. Immunol. 155:3946 (1995)),growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT; Roskam et al., NucleicAcids Res. 7:30 (1979)); preproinsulin (MALWMRLLPLLALLALWGPOPAAAFVN;Bell et al., Nature 284:26 (1980)); and influenza HA protein(MKAKLLVLLYAFVAGDQI; Sekiwawa et al., PNAS 80:3563)), with cleavagebetween the non-underlined-underlined junction. A particularly preferredsecretory signal sequence is the signal leader sequence from thesecreted cytokine IL-4, which comprises the first 24 amino acids of IL-4as follows: MGLTSQLLPPLFFLLACAGNFVHG.

In a preferred embodiment, the fusion partner is a rescue sequence. Arescue sequence is a sequence which may be used to purify or isolateeither the peptide or the nucleic acid encoding it. Thus, for example,peptide rescue sequences include purification sequences such as the His₆tag for use with Ni affinity columns and epitope tags for detection,immunoprecipitation or FACS (fluorescence-activated cell sorting).Suitable epitope tags include myc (for use with the commerciallyavailable 9E10 antibody), the BSP biotinylation target sequence of thebacterial enzyme BirA, flu tags, lacZ, GST, and Strep tag I and II.

Alternatively, the rescue sequence may be a unique oligonucleotidesequence which serves as a probe target site to allow the quick and easyisolation of the retroviral construct, via PCR, related techniques, orhybridization.

While the discussion has been directed to the fusion of fusion partnersto the intein portion of the fusion polypeptide, the fusion partners maybe placed anywhere (i.e. N-terminal, C-terminal, internal) in thestructure as the biology and activity permits. In addition, it is alsopossible to fuse one or more of these fusion partners to the inteinportions of the fusion polypeptide. Thus, for example, a targetingsequence (either N-terminally, C-terminally, or internally, as describedbelow) may be fused to intein A, and a rescue sequence fused to the sameplace or a different place on the molecule. Thus, any combination offusion partners and peptides may be made.

In a preferred embodiment, the invention provides libraries of fusionpolypeptides. By “library” herein is meant a sufficiently structurallydiverse population of randomized expression products to effect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Accordingly, an interactionlibrary must be large enough so that at least one of its members willhave a structure that gives it affinity for some molecule, protein, orother factor whose activity is of interest. Although it is difficult togauge the required absolute size of an interaction library, natureprovides a hint with the immune response: a diversity of 10⁷-10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.Published in vitro selection techniques have also shown that a librarysize of 10⁷ to 10⁸ is sufficient to find structures with affinity forthe target. A library of all combinations of a peptide 7 to 20 aminoacids in length, such as proposed here for expression in retroviruses,has the potential to code for 20⁷ (10⁹) to 20²⁰. Thus, with libraries of10⁷ to 10⁸ per ml of retroviral particles the present methods allow a“working” subset of a theoretically complete interaction library for 7amino acids, and a subset of shapes for the 20²⁰ library. Thus, in apreferred embodiment, at least 10⁶, preferably at least 10⁷, morepreferably at least 10⁸ and most preferably at least 10⁹ differentexpression products are simultaneously analyzed in the subject methods.Preferred methods maximize library size and diversity.

In a preferred embodiment, libraries of all combinations of a peptide 3to 30 amino acids in length are synthesized and analyzed as outlinedherein. Libraries of smaller cyclic peptides, i.e., 3 to 4 amino acid inlength, are advantageous because they are more constrained and thusthere is a better chance that these libraries possess desirablepharmocokinetics properties as a consequence of their smaller size.Accordingly, the libraries of the present invention may be one of any ofthe following lengths: 3 amino acids, 4 amino acids, 5 amino acids, 6amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 aminoacids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids,15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 aminoacids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids,24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 aminoacids, 29 amino acids and 30 amino acids in length.

The invention further provides fusion nucleic acids encoding the fusionpolypeptides of the invention. As will be appreciated by those in theart, due to the degeneracy of the genetic code, an extremely largenumber of nucleic acids may be made, all of which encode the fusionproteins of the present invention. Thus, having identified a particularamino acid sequence, those skilled in the art could make any number ofdifferent nucleic acids, by simply modifying the sequence of one or morecodons in a way which does not change the amino acid sequence of thefusion protein.

Using the nucleic acids of the present invention which encode a fusionprotein, a variety of expression vectors are made. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genome. Generally, these expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the fusion protein.The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

The fusion nucleic acids are introduced into cells to screen for cyclicpeptides capable of altering the phenotype of a cell. By “introducedinto” or grammatical equivalents herein is meant that the nucleic acidsenter the cells in a manner suitable for subsequent expression of thenucleic acid. The method of introduction is largely dictated by thetargeted cell type, discussed below. Exemplary methods include CaPO₄precipitation, liposome fusion, lipofectin®, electroporation, viralinfection, etc. The fusion nucleic acids may stably integrate into thegenome of the host cell (for example, with retroviral introduction,outlined below), or may exist either transiently or stably in thecytoplasm (i.e. through the use of traditional plasmids, utilizingstandard regulatory sequences, selection markers, etc.). As manypharmaceutically important screens require human or model mammalian celltargets, retroviral vectors capable of transfecting such targets arepreferred.

In a preferred embodiment, the fusion nucleic acids are part of aretroviral particle which infects the cells. Generally, infection of thecells is straightforward with the application of the infection-enhancingreagent polybrene, which is a polycation that facilitates viral bindingto the target cell. Infection can be optimized such that each cellgenerally expresses a single construct, using the ratio of virusparticles to number of cells. Infection follows a Poisson distribution.

In a preferred embodiment, the fusion nucleic acids are introduced intocells using retroviral vectors. Currently, the most efficient genetransfer methodologies harness the capacity of engineered viruses, suchas retroviruses, to bypass natural cellular barriers to exogenousnucleic acid uptake. The use of recombinant retroviruses was pioneeredby Richard Mulligan and David Baltimore with the Psi-2 lines andanalogous retrovirus packaging systems, based on NIH 3T3 cells (see Mannet al., Cell 33:153-159 (1993), hereby incorporated by reference). Suchhelper-defective packaging lines are capable of producing all thenecessary trans proteins-gag, pol, and env- that are required forpackaging, processing, reverse transcription, and integration ofrecombinant genomes. Those RNA molecules that have in cis the Ψpackaging signal are packaged into maturing virions. Retroviruses arepreferred for a number of reasons. First, their derivation is easy.Second, unlike Adenovirus-mediated gene delivery, expression fromretroviruses is long-term (adenoviruses do not integrate).Adeno-associated viruses have limited space for genes and regulatoryunits and there is some controversy as to their ability to integrate.Retroviruses therefore offer the best current compromise in terms oflong-term expression, genomic flexibility, and stable integration, amongother features. The main advantage of retroviruses is that theirintegration into the host genome allows for their stable transmissionthrough cell division. This ensures that in cell types which undergomultiple independent maturation steps, such as hematopoietic cellprogression, the retrovirus construct will remain resident and continueto express.

A particularly well suited retroviral transfection system is describedin Mann et al., supra: Pear et al., PNAS USA 90(18):8392-6 (1993);Kitamura et al., PNAS USA 92:9146-9150 (1995); Kinsella et al., HumanGene Therapy 7:1405-1413; Hofmann et al., PNAS USA 93:5185-5190; Choateet al., Human Gene Therapy 7:2247 (1996); and WO 94/19478; andreferences cited therein, all of which are incorporated by reference.

In one embodiment of the invention, the library is generated in aintein-catalyzed cyclization scaffold. By “intein-catalyzed cyclizationscaffold” herein is meant that the intein is engineered such that acyclic peptide is generated upon intein-mediated splicing of theextein-intein junction points. Preferably, an intein cyclizationscaffold includes the C-terminal intein motif, a library insert of 3 upto 30 amino acids in length, and the N-terminal intein motif. The C- andN-terminal intein motifs can be derived from any number of known inteinscapable mediating protein splicing, including split-inteins.

Most wild-type inteins have requirements for a specific extein-encodedamino acid at the C-intein (IntB)/C-extein junction point. This variesdepending on the intein, but most often consists of an cysteine,threonine or serine. Intein-generated cyclic peptide libraries may begenerated in which this particular amino acid is fixed and correspondsto the amino acid present in the wild-type sequence. For example, theSsp. DnaB intein utilizes an extein-encoded serine in this position.

A number of inteins have the ability to catalyze protein splicing whennon-native amino acids are substituted at the C-intein (IntB)/C-exteinjunction point position. Degeneracy at the C-intein (IntB)/C-exteinjunction point position leads to cyclic peptide libraries of greatercomplexity and therefore added utility. The proposed degeneracy in thisposition most likely consists of a cysteine, serine or threonine but isnot limited to these amino acids. The ability of a givenintein-catalyzed cyclization scaffold to tolerate degeneracy at thisposition depends on the specific intein utilized and its mechanism ofprotein splicing. Thus, isolation of intein cyclization scaffolds with agreater tolerance for degeneracy at the C-intein (IntB)/C-exteinjunction point is within the scope of this invention.

In one embodiment of the invention, the library is generated in aretrovirus DNA construct backbone, as is generally described in U.S.Ser. No. 08/789,333, filed Jan. 23, 1997, incorporated herein byreference. Standard oligonucleotide synthesis is done to generate therandom portion of the candidate bioactive agent, using techniques wellknown in the art (see Eckstein, Oligonucleotides and Analogues, APractical Approach, IRL Press at Oxford University Press, 1991);libraries may be commercially purchased. Libraries with up to 10⁹ to10¹⁰ unique sequences can be readily generated in such DNA backbones.After generation of the DNA library, the library is cloned into a firstprimer. The first primer serves as a “cassette”, which is inserted intothe retroviral construct The first primer generally contains a number ofelements, including for example, the required regulatory sequences (e.g.translation, transcription, promoters, etc), fusion partners,restriction endonuclease (cloning and subcloning) sites, stop codons(preferably in all three frames), regions of complementarity for secondstrand priming (preferably at the end of the stop codon region as minordeletions or insertions may occur in the random region), etc. See U.S.Ser. No. 08/789,333, hereby incorporated by reference.

A second primer is then added, which generally consists of some or allof the complementarity region to prime the first primer and optionalnecessary sequences for a second unique restriction site for subcloning.DNA polymerase is added to make double-stranded oligonucleotides. Thedouble-stranded oligonucleotides are cleaved with the appropriatesubcloning restriction endonucleases and subcloned into the targetretroviral vectors, described below.

Any number of suitable retroviral vectors may be used. Generally, theretroviral vectors may include: selectable marker genes under thecontrol of internal ribosome entry sites (IRES), which allows forbicistronic operons and thus greatly facilitates the selection of cellsexpressing peptides at uniformly high levels; and promoters drivingexpression of a second gene, placed in sense or anti-sense relative tothe 5′ LTR. Suitable selection genes include, but are not limited to,neomycin, blastocidin, bleomycin, puromycin, and hygromycin resistancegenes, as well as self-fluorescent markers such as green fluorescentprotein, enzymatic markers such as lacZ, and surface proteins such asCD8, etc.

Preferred vectors include a vector based on the murine stem cell virus(MSCV) (see Hawley et al., Gene Therapy 1:136 (1994)) and a modified MFGvirus (Rivere et al., Genetics 92:6733 (1995)), and pBABE, outlined inthe examples. A general schematic of the retroviral construct isdepicted in FIG. 6.

The retroviruses may include inducible and constitutive promoters. Forexample, there are situations wherein it is necessary to induce peptideexpression only during certain phases of the selection process. Forinstance, a scheme to provide pro-inflammatory cytokines in certaininstances must include induced expression of the peptides. This isbecause there is some expectation that over-expressed pro-inflammatorydrugs might in the long-term be detrimental to cell growth. Accordingly,constitutive expression is undesirable, and the peptide is only turnedon during that phase of the selection process when the phenotype isrequired, and then shut the peptide down by turning off the retroviralexpression to confirm the effect or ensure long-term survival of theproducer cells. A large number of both inducible and constitutivepromoters are known.

In addition, it is possible to configure a retroviral vector to allowinducible expression of retroviral inserts after integration of a singlevector in target cells; importantly, the entire system is containedwithin the single retrovirus. Tet-inducible retroviruses have beendesigned incorporating the Self-Inactivating (SIN) feature of 3′ LTRenhancer/promoter retroviral deletion mutant (Hoffman et al., PNAS USA93:5185 (1996)). Expression of this vector in cells is virtuallyundetectable in the presence of tetracycline or other active analogs.However, in the absence of Tet, expression is turned on to maximumwithin 48 hours after induction, with uniform increased expression ofthe whole population of cells that harbor the inducible retrovirus,indicating that expression is regulated uniformly within the infectedcell population. A similar, related system uses a mutated TetDNA-binding domain such that it bound DNA in the presence of Tet, andwas removed in the absence of Tet. Either of these systems is suitable.

In this manner the primers create a library of fragments, eachcontaining a different random nucleotide sequence that may encode adifferent peptide. The ligation products are then transformed intobacteria, such as E. coli, and DNA is prepared from the resultinglibrary, as is generally outlined in Kitamura, PNAS USA 92:9146-9150(1995), hereby expressly incorporated by reference.

Delivery of the library DNA into a retroviral packaging system resultsin conversion to infectious virus. Suitable retroviral packaging systemcell lines include, but are not limited to, the Bing and BOSC23 celllines described in WO 94/19478; Soneoka et al., Nucleic Acid Res.23(4):628 (1995); Finer et al., Blood 83:43 (1994); Pheonix packaginglines such as PhiNX-eco and PhiNX-ampho, described below; 292T+gag-poland retrovirus envelope; PA317; and cell lines outlined in Markowitz etal., Virology 167:400 (1988), Markowitz et al., J. Virol. 62:1120(1988), Li et al., PNAS USA 93:11658 (1996), Kinsella et al., Human GeneTherapy 7:1405 (1996), all of which are incorporated by reference.

Preferred systems include PHEONIX-ECO and PHEONIX-AMPHO. BothPHEONIX-ECO and PHEONIX-AMPHO were tested for helper virus productionand established as being helper-virus free. Both lines can carryepisomes for the creation of stable cell lines which can be used toproduce retrovirus. Both lines are readily testable by flow cytometryfor stability of gag-pol (CD8) and envelope expression; after severalmonths of testing the lines appear stable, and do not demonstrate lossof titre as did the first-generation lines BOSC23 and Bing (partly dueto the choice of promoters driving expression of gag-pol and envelope).Both lines can also be used to transiently produce virus in a few days.Thus, these lines are fully compatible with transient, episomal stable,and library generation for retroviral gene transfer experiments.Finally, the titres produced by these lines have been tested. Usingstandard polybrene-enhanced retroviral infection, titres approaching orabove 10⁷ per ml were observed for both PHEONIX-eco and PHEONIX-amphowhen carrying episomal constructs. When transiently produced virus ismade, titres are usually ½ to ⅓ that value.

These lines are helper-virus free, carry episomes for long-term stableproduction of retrovirus, stably produce gag-pol and env, and do notdemonstrate loss of viral titre over time. In addition, PhiNX-eco andPhiNX-ampho are capable of producing titres approaching or above 10⁷ perml when carrying episomal constructs, which, with concentration ofvirus, can be enhanced to 10⁸ to 10⁹ per ml.

In a preferred embodiment, the cell lines disclosed above, and the othermethods for producing retrovirus, are useful for production of virus bytransient transfection. The virus can either be used directly or be usedto infect another retroviral producer cell line for “expansion” of thelibrary.

Concentration of virus may be done as follows. Generally, retrovirusesare titred by applying retrovirus-containing supernatant onto indicatorcells, such as NIH3T3 cells, and then measuring the percentage of cellsexpressing phenotypic consequences of infection. The concentration ofthe virus is determined by multiplying the percentage of cells infectedby the dilution factor involved, and taking into account the number oftarget cells available to obtain a relative titre. If the retroviruscontains a reporter gene, such as lacZ, then infection, integration, andexpression of the recombinant virus is measured by histological stainingfor lacZ expression or by flow cytometry (FACS). In general, retroviraltitres generated from even the best of the producer cells do not exceed10⁷ per ml, unless concentration by relatively expensive or exoticapparatus. However, as it has been recently postulated that since aparticle as large as a retrovirus will not move very far by brownianmotion in liquid, fluid dynamics predicts that much of the virus nevercomes in contact with the cells to initiate the infection process.However, if cells are grown or placed on a porous filter and retrovirusis allowed to move past cells by gradual gravitometric flow, a highconcentration of virus around cells can be effectively maintained at alltimes. Thus, up to a ten-fold higher infectivity by infecting cells on aporous membrane and allowing retrovirus supernatant to flow past themhas been seen. This should allow titres of 10⁹ after concentration.

The fusion nucleic acids and polypeptides of the invention are used tomake cyclic peptides. By “cyclic peptides” or grammatical equivalentsherein is meant the intracellular catalysis of peptide backbonecyclization. Generally, backbone cyclization results in the joining ofthe N and C termini of a peptide together such that a cyclic product isgenerated inside cells.

Preferably, every member of a peptide library is tested for bioactivityusing one of the assays described below. For example, a cyclic peptidewith 7 random positions has a complexity of 20⁷=1.28×10⁹, all of whichwill be tested for biological activity.

In the event it is not possible to test every member of a library forbioactivity, the library may be deliberately biased. For example, acyclic peptide can be biased to cellular entry by fixing one or morerelatively hydrophobic amino acids, such as tyrosine or tryptophan.Other types of biased libraries which may be synthesized includelibraries which primary contain cyclic peptides comprising amino acidswith large side chains and libraries in which the number of cyclicpeptide conformers is restricted.

Highly restrained cyclic peptide libraries are made by using codonswhich code mainly for amino acids with large side chains. That is, whenseveral resides of a cyclic peptide encode amino acids with large sidechains, the conformation space of the peptide is restricted. The resultis to bias the peptide to a higher affinity by reducing peptideconformational entropy. For example, a library of cyclic peptides couldbe created by restricting the triplet nucleotides coding for each randomamino acid in the library to C or T for the first position of thetriplet, A, G or T for the second position in the triplet, and G, C or Tfor the third position in the triplet. This would result in a librarybiased to large amino acids, i.e., phenylalanine (F), leucine (L),tyrosine (Y), histidine (H), glutamine (Q), cysteine (C), tryptophan (W)and arginine (R). A library biased toward large amino acid side chains,combined with the loss of glycine, alanine, serine, threonine,aspartate, and glutamate results in a library coding for more rigidpeptides. As this library lacks an acidic amino acid, a pre-synthesizedtriplet coding glutamate (i.e., GAG) or aspartate (GAC) may be addedduring the DNA synthesis of the library.

Alternatively, a large amino acid side chain (i.e.) residue library maybe created by pre-synthesizing triplets for desired residues. Theseresidues are then mixed together during the DNA synthesis of thelibrary. An example of a pre-synthesized large residue library is alibrary coding tyrosine (Y), arginine (R), glutamic acid (E), histidine(H), leucine (L), glutamine (Q), and optionally proline (P) or threonine(T).

A biased library can be created by restricting the number of conformersin a cyclic peptide. This approach is useful for structure activityrelationship optimization. The number of conformers may be restricted byfixing a proline in the cyclic peptide ring at one position and leavingall of the other residues random. A smaller number of conformers allowsfor higher affinity binding interactions with target molecules, and moreselective interactions with target molecules due to a diminution of thepossibility of “induced fit” binding. “Induced fit” comes at the expenseof binding affinity due to a loss upon binding of the higherconformational entropy of a multi-conformer peptide. Higher affinity andselectivity are desirable for the development of cyclic peptides drugs.This is achieved by reducing the conformational entropy by including arigid amino acid in a fixed position in each library member. Forexample, fixing one proline in a 7mer peptide is sufficient to restrictthe conformational space of the cyclic peptide. For 8 to 10 mers, twoprolines may be fixed in the ring allowing a diversity of (20)⁶ or6.4×10⁷ in the 6 unfixed position of a 10 mer ring. Such a library islarge enough to give hits in most screens for candidate drugs (asdescribed below).

As will be appreciated by those in the art, the type of cells used inthe present invention can vary widely. Basically, any mammalian cellsmay be used, with mouse, rat, primate and human cells being particularlypreferred, although as will be appreciated by those in the art,modifications of the system by pseudotyping allows all eukaryotic cellsto be used, preferably higher eukaryotes. As is more fully describedbelow, a screen will be set up such that the cells exhibit a selectablephenotype in the presence of a cyclic peptide. As is more fullydescribed below, cell types implicated in a wide variety of diseaseconditions are particularly useful, so long as a suitable screen may bedesigned to allow the selection of cells that exhibit an alteredphenotype as a consequence of the presence of a cyclic peptide withinthe cell.

Accordingly, suitable cell types include, but are not limited to, tumorcells of all types (particularly melanoma, myeloid leukemia, carcinomasof the lung, breast, ovaries, colon, kidney, prostate, pancreas andtestes), cardiomyocytes, endothelial cells, epithelial cells,lymphocytes (T-cell and B cell), mast cells, eosinophils, vascularintimal cells, hepatocytes, leukocytes including mononuclear leukocytes,stem cells such as haemopoetic, neural, skin, lung, kidney, liver andmyocyte stem cells (for use in screening for differentiation andde-differentiation factors), osteoclasts, chondrocytes and otherconnective tissue cells, keratinocytes, melanocytes, liver cells, kidneycells, and adipocytes. Suitable cells also include known research cells,including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos,etc. See the ATCC cell line catalog, hereby expressly incorporated byreference.

In one embodiment, the cells may be genetically engineered, that is,contain exogenous nucleic acid, for example, to contain targetmolecules.

Once made, the compositions of the invention find use in a number ofapplications. In particular, compositions with altered cyclizationefficiency are made. The compositions of the invention also may be usedto: (1) alter cellular phenotypes and/or physiology; (2) used inscreening assays to identify target molecules associated with changes incellular phenotype or physiology; and, (3) used as drugs to treat anumber of disease states, such as cancer, cardiovascular diseases,obesity, neurological disorders, etc.

In a preferred embodiment, inteins with altered cyclization activity aregenerated. Naturally occurring inteins are mutagenized and tested invivo to determine whether the modified intein can catalyze protein orpeptide cyclization in mammalian cells. Preferably, inteins so modifiedare characterized by more efficient cyclization kinetics in vivo or bythe expression level of intein catalyzed cyclization scaffolds.Additional rounds of mutagenesis may be done to optimize in vivofunction. Assays useful for measuring intein-catalyzed cyclizationefficiency include fluorescent or gel based assays directly measuringcyclic peptide or protein levels, and functional assays based on theproduction of a functional cyclic peptide whose effects can be measuredor selected for.

In a preferred embodiment, random mutagenesis (i.e. M13 primermutagenesis and PCR mutagenesis), PCR shuffling or other directedevolution techniques are directed to a target codon or region and theresulting intein variants screened for altered cyclization activity.These techniques are well known and can be directed to predeterminedsites, i.e., intein open reading frame or more specific regions orcodons within.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the intein protein aredesired, substitutions are generally made in accordance with thefollowing chart: CHART I Original Residue Exemplary Substitutions AlaSer Arg Lys Asn Gln, His Asp Glu Cys Ser Gin Asn Glu Asp Gly Pro HisAsn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile PheMet, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function are made by selecting substitutions thatare less conservative than those shown in Chart I. For example,substitutions may be made which more significantly affect: the structureof the polypeptide backbone in the area of the alteration, for examplethe alpha-helical or beta-sheet structure; the charge or hydrophobicityof the molecule at the target site; or the bulk of the side chain. Thesubstitutions which in general are expected to produce the greatestchanges in the polypeptide's properties are those in which (a) ahydrophilic residue, e.g. seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g. lysyl,arginyl, or histidyl, is substituted for (or by) an electronegativeresidue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky sidechain, e.g. phenylalanine, is substituted for (or by) one not having aside chain, e.g. glycine.

As outlined above, the variants typically exhibit the same qualitativebiological activity (i.e. cyclization) although variants may be selectedto modify other characteristics of the intein protein as needed. Forexample, endoplasmic reticulum/golgi directed intein libraries may bedesigned to operate in cellular environments more acidic than thecytoplasmic compartment.

In a preferred embodiment specific residues of an intein motif aresubstituted, resulting in proteins with modified characteristics. Suchsubstitutions may occur at one or more residues, with 1-10 substitutionsbeing preferred. Preferred characteristics to be modified includecyclization efficiency, half-life, stability, temperature sensitivity.

In a preferred embodiment, intein mutants are generated using PCRmutagenesis. The resulting mutants are screened for altered cyclizationactivity. By “altered” cyclization activity” refers to anycharacteristic or attribute of an intein that can be selected ordetected and compared to the corresponding property of a naturallyoccurring intein. These properties include cyclization efficiency,stability, etc. Cyclization efficiency may be affected by the presenceor absence of a given amino acid, the size of the peptide library, etc.

Unless otherwise specified, altered” cyclization activity, whencomparing the cyclization efficiency of a mutant intein to thecyclization efficiency of wild-type or naturally occurring intein ispreferably at least 1-fold, more preferably at least a 10-fold increasein activity.

Screens for mutants with improved cyclization efficiency can be done inprocaryotes or eucaryotes. The mutants may be screened directly byassaying for the production of a cyclic peptide or indirectly byassaying a cyclic peptide's effects on a cell. Alternatively, themutants may be screened indirectly by assaying the product of the cyclicpeptide protein in vitro, e.g., enzyme inhibition assays, etc.

If the mutation prevents self-excision, no fluorescence is detected dueto the interruption in the tertiary structure of GFP. If the mutationdoes not effect self-excision or enhances splicing efficiency, thedegree of fluorescence may be quantified using a FACS analysis or othertechniques known in the art. In addition, cyclization of the GFPreconstitutes the myc epitope which can be detected using Westernanalysis. T

In a preferred embodiment, a first plurality of cells is screened. Thatis, the cells into which the fusion nucleic acids are introduced arescreened for an altered phenotype. Thus, in this embodiment, the effectof the bioactive peptide is seen in the same cells in which it is made;i.e. an autocrine effect.

By a “plurality of cells” herein is meant roughly from about 10³ cellsto 10⁸ or 10⁹, with from 10⁶ to 10⁸ being preferred. This plurality ofcells comprises a cellular library, wherein generally each cell withinthe library contains a member of the peptide molecular library, i.e. adifferent peptide (or nucleic acid encoding the peptide), although aswill be appreciated by those in the art, some cells within the librarymay not contain a peptide, and some may contain more than species ofpeptide. When methods other than retroviral infection are used tointroduce the candidate nucleic acids into a plurality of cells, thedistribution of candidate nucleic acids within the individual cellmembers of the cellular library may vary widely, as it is generallydifficult to control the number of nucleic acids which enter a cellduring electroporation, etc.

In a preferred embodiment, the fusion nucleic acids are introduced intoa first plurality of cells, and the effect of the peptide is screened ina second or third plurality of cells, different from the first pluralityof cells, i.e. generally a different cell type. That is, the effect ofthe bioactive peptide is due to an extracellular effect on a secondcell; i.e. an endocrine or paracrine effect. This is done using standardtechniques. The first plurality of cells may be grown in or on onemedia, and the media is allowed to touch a second plurality of cells,and the effect measured. Alternatively, there may be direct contactbetween the cells. Thus, “contacting” is functional contact, andincludes both direct and indirect. In this embodiment, the firstplurality of cells may or may not be screened.

If necessary, the cells are treated to conditions suitable for theexpression of the peptide (for example, when inducible promoters areused).

Thus, the methods of the present invention comprise introducing amolecular library of fusion nucleic acids encoding randomized peptidesfused to scaffold into a plurality of cells, a cellular library. Each ofthe nucleic acids comprises a different nucleotide sequence encodingscaffold with a random peptide. The plurality of cells is then screened,as is more fully outlined below, for a cell exhibiting an alteredphenotype. The altered phenotype is due to the presence of a bioactivepeptide.

By “altered phenotype” or “changed physiology” or other grammaticalequivalents herein is meant that the phenotype of the cell is altered insome way, preferably in some detectable and/or measurable way. As willbe appreciated in the art, a strength of the present invention is thewide variety of cell types and potential phenotypic changes which may betested using the present methods. Accordingly, any phenotypic changewhich may be observed, detected, or measured may be the basis of thescreening methods herein. Suitable phenotypic changes include, but arenot limited to: gross physical changes such as changes in cellmorphology, cell growth, cell viability, adhesion to substrates or othercells, and cellular density; changes in the expression of one or moreRNAs, proteins, lipids, hormones, cytokines, or other molecules; changesin the equilibrium state (i.e. half-life) or one or more RNAs, proteins,lipids, hormones, cytokines, or other molecules; changes in thelocalization of one or more RNAs, proteins, lipids, hormones, cytokines,or other molecules; changes in the bioactivity or specific activity ofone or more RNAs, proteins, lipids, hormones, cytokines, receptors, orother molecules; changes in the secretion of ions, cytokines, hormones,growth factors, or other molecules; alterations in cellular membranepotentials, polarization, integrity or transport; changes ininfectivity, susceptability, latency, adhesion, and uptake of virusesand bacterial pathogens; etc. By “capable of altering the phenotype”herein is meant that the bioactive peptide can change the phenotype ofthe cell in some detectable and/or measurable way.

The altered phenotype may be detected in a wide variety of ways, as isdescribed more fully below, and will generally depend and correspond tothe phenotype that is being changed. Generally, the changed phenotype isdetected using, for example: microscopic analysis of cell morphology;standard cell viability assays, including both increased cell death andincreased cell viability, for example, cells that are now resistant tocell death via virus, bacteria, or bacterial or synthetic toxins;standard labeling assays such as fluorometric indicator assays for thepresence or level of a particular cell or molecule, including FACS orother dye staining techniques; biochemical detection of the expressionof target compounds after killing the cells; etc. In some cases, as ismore fully described herein, the altered phenotype is detected in thecell in which the fusion nucleic acid was introduced; in otherembodiments, the altered phenotype is detected in a second cell which isresponding to some molecular signal from the first cell.

An altered phenotype of a cell indicates the presence of a bioactivepeptide, acting preferably in a transdominant way. By “transdominant”herein is meant that the bioactive peptide indirectly causes the alteredphenotype by acting on a second molecule, which leads to an alteredphenotype. That is, a transdominant expression product has an effectthat is not in cis, i.e., a trans event as defined in genetic terms orbiochemical terms. A transdominant effect is a distinguishable effect bya molecular entity (i.e., the encoded peptide or RNA) upon some separateand distinguishable target; that is, not an effect upon the encodedentity itself. As such, transdominant effects include many well-knowneffects by pharmacologic agents upon target molecules or pathways incells or physiologic systems; for instance, the β-lactam antibioticshave a transdominant effect upon peptidoglycan synthesis in bacterialcells by binding to penicillin binding proteins and disrupting theirfunctions. An exemplary transdominant effect by a peptide is the abilityto inhibit NF-κB signaling by binding to IκB-α at a region critical forits function, such that in the presence of sufficient amounts of thepeptide (or molecular entity), the signaling pathways that normally leadto the activation of NF-κB through phosphorylation and/or degradation ofIκB-α are inhibited from acting at IκB-α because of the binding of thepeptide or molecular entity. In another instance, signaling pathwaysthat are normally activated to secrete IgE are inhibited in the presenceof peptide. Or, signaling pathways in adipose tissue cells, normallyquiescent, are activated to metabolize fat. Or, in the presence of apeptide, intracellular mechanisms for the replication of certainviruses, such as HIV-I, or Herpes viridae family members, or RespiratorySyncytia Virus, for example, are inhibited.

A transdominant effect upon a protein or molecular pathway is clearlydistinguishable from randomization, change, or mutation of a sequencewithin a protein or molecule of known or unknown function to enhance ordiminish a biochemical ability that protein or molecule alreadymanifests. For instance, a protein that enzymatically cleaves β-lactamantibiotics, a β-lactamase, could be enhanced or diminished in itsactivity by mutating sequences internal to its structure that enhance ordiminish the ability of this enzyme to act upon and cleave β-lactamantibiotics. This would be called a cis mutation to the protein. Theeffect of this protein upon β-lactam antibiotics is an activity theprotein already manifests, to a distinguishable degree. Similarly, amutation in the leader sequence that enhanced the export of this proteinto the extracellular spaces wherein it might encounter β-lactammolecules more readily, or a mutation within the sequence that enhancethe stability of the protein, would be termed cis mutations in theprotein. For comparison, a transdominant effector of this protein wouldinclude an agent, independent of the β-lactamase, that bound to theβ-lactamase in such a way that it enhanced or diminished the function ofthe β-lactamase by virtue of its binding to β-lactamase.

In a preferred embodiment, once a cell with an altered phenotype isdetected, the presence of the fusion protein is verified, to ensure thatthe peptide was expressed and thus that the altered phenotype can be dueto the presence of the peptide. As will be appreciated by those in theart, this verification of the presence of the peptide can be done eitherbefore, during or after the screening for an altered phenotype. This canbe done in a variety of ways, although preferred methods utilize FACStechniques.

In a preferred embodiment, the devices of the invention comprise liquidhandling components, including components for loading and unloadingfluids at each station or sets of stations. The liquid handling systemscan include robotic systems comprising any number of components. Inaddition, any or all of the steps outlined herein may be automated;thus, for example, the systems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which can be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates; holderswith cartridges and/or caps; automated lid or cap handlers to remove andreplace lids for wells on non-cross contamination plates; tip assembliesfor sample distribution with disposable tips; washable tip assembliesfor sample distribution; 96 well loading blocks; cooled reagent racks;microtitler plate pipette positions (optionally cooled); stacking towersfor plates and tips; and computer systems.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting to perform all steps of screening applications. This includesliquid, particle, cell, and organism manipulations such as aspiration,dispensing, mixing, diluting, washing, accurate volumetric transfers;retrieving, and discarding of pipet tips; and repetitive pipetting ofidentical volumes for multiple deliveries from a single sampleaspiration. These manipulations are cross-contamination-free liquid,particle, cell, and organism transfers. This instrument performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and high capacity operation.

In a preferred embodiment, chemically derivatized particles, plates,cartridges, tubes, magnetic particles, or other solid phase matrix withspecificity to the assay components are used. The binding surfaces ofmicroplates, tubes or any solid phase matrices include non-polarsurfaces, highly polar surfaces, modified dextran coating to promotecovalent binding, antibody coating, affinity media to bind fusionproteins or peptides, surface-fixed proteins such as recombinant proteinA or G, nucleotide resins or coatings, and other affinity matrix areuseful in this invention.

In a preferred embodiment, platforms for multi-well plates, multi-tubes,holders, cartridges, minitubes, deep-well plates, microfuge tubes,cryovials, square well plates, filters, chips, optic fibers, beads, andother solid-phase matrices or platform with various volumes areaccommodated on an upgradable modular platform for additional capacity.This modular platform includes a variable speed orbital shaker, andmulti-position work decks for source samples, sample and reagentdilution, assay plates, sample and reagent reservoirs, pipette tips, andan active wash station.

In a preferred embodiment, thermocycler and thermoregulating systems areused for stabilizing the temperature of the heat exchangers such ascontrolled blocks or platforms to provide accurate temperature controlof incubating samples from 4° C. to 100° C.; this is in addition to orin place of the station thermocontrollers.

In a preferred embodiment, interchangeable pipet heads (single ormulti-channel) with single or multiple magnetic probes, affinity probes,or pipetters robotically manipulate the liquid, particles, cells, andorganisms. Multi-well or multi-tube magnetic separators or platformsmanipulate liquid, particles, cells, and organisms in single or multiplesample formats.

In some embodiments, for example when electronic detection is not done,the instrumentation will include a detector, which can be a wide varietyof different detectors, depending on the labels and assay. In apreferred embodiment, useful detectors include a microscope(s) withmultiple channels of fluorescence; plate readers to provide fluorescent,ultraviolet and visible spectrophotometric detection with single anddual wavelength endpoint and kinetics capability, fluroescence resonanceenergy transfer (FRET), luminescence, quenching, two-photon excitation,and intensity redistribution; CCD cameras to capture and transform dataand images into quantifiable formats; and a computer workstation.

These instruments can fit in a sterile laminar flow or fume hood, or areenclosed, self-contained systems, for cell culture growth andtransformation in multi-well plates or tubes and for hazardousoperations. The living cells will be grown under controlled growthconditions, with controls for temperature, humidity, and gas for timeseries of the live cell assays. Automated transformation of cells andautomated colony pickers will facilitate rapid screening of desiredcells.

Flow cytometry or capillary electrophoresis formats can be used forindividual capture of magnetic and other beads, particles, cells, andorganisms.

The flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.The customized tools, labware, and liquid, particle, cell and organismtransfer patterns allow different applications to be performed. Thedatabase allows method and parameter storage. Robotic and computerinterfaces allow communication between instruments.

In a preferred embodiment, the robotic apparatus includes a centralprocessing unit which communicates with a memory and a set ofinput/output devices (e.g., keyboard, mouse, monitor, printer, etc.)through a bus. Again, as outlined below, this may be in addition to orin place of the CPU for the multiplexing devices of the invention. Thegeneral interaction between a central processing unit, a memory,input/output devices, and a bus is known in the art. Thus, a variety ofdifferent procedures, depending on the experiments to be run, are storedin the CPU memory.

These robotic fluid handling systems can utilize any number of differentreagents, including buffers, reagents, samples, washes, assay componentssuch as label probes, etc.

Once the presence of the fusion protein is verified, the cell with thealtered phenotype is generally isolated from the plurality which do nothave altered phenotypes. This may be done in any number of ways, as isknown in the art, and will in some instances depend on the assay orscreen. Suitable isolation techniques include, but are not limited to,FACS, lysis selection using complement, cell cloning, scanning byFluorimager, expression of a “survival” protein, induced expression of acell surface protein or other molecule that can be rendered fluorescentor taggable for physical isolation; expression of an enzyme that changesa non-fluorescent molecule to a fluorescent one; overgrowth against abackground of no or slow growth; death of cells and isolation of DNA orother cell vitality indicator dyes, etc.

In a preferred embodiment, the fusion nucleic acid and/or the bioactivepeptide (i.e. the fusion protein) is isolated from the positive cell.This may be done in a number of ways. In a preferred embodiment, primerscomplementary to DNA regions common to the retroviral constructs, or tospecific components of the library such as a rescue sequence, definedabove, are used to “rescue” the unique random sequence. Alternatively,the fusion protein is isolated using a rescue sequence. Thus, forexample, rescue sequences comprising epitope tags or purificationsequences may be used to pull out the fusion protein usingimmunoprecipitation or affinity columns. In some instances, as isoutlined below, this may also pull out the primary target molecule, ifthere is a sufficiently strong binding interaction between the bioactivepeptide and the target molecule. Alternatively, the peptide may bedetected using mass spectroscopy.

Once rescued, the sequence of the bioactive peptide and/or fusionnucleic acid is determined. This information can then be used in anumber of ways.

In a preferred embodiment, the bioactive peptide is resynthesized andreintroduced into the target cells, to verify the effect. This may bedone using retroviruses, or alternatively using fusions to the HIV-1 Tatprotein, and analogs and related proteins, which allows very high uptakeinto target cells. See for example, Fawell et al., PNAS USA 91:664(1994); Frankel et al., Cell 55:1189 (1988); Savion et al., J. Biol.Chem. 256:1149 (1981); Derossi et al., J. Biol. Chem. 269:10444 (1994);and Baldin et al., EMBO J. 9:1511 (1990), all of which are incorporatedby reference.

In a preferred embodiment, the sequence of a bioactive peptide is usedto generate more candidate peptides. For example, the sequence of thebioactive peptide may be the basis of a second round of (biased)randomization, to develop bioactive peptides with increased or alteredactivities. Alternatively, the second round of randomization may changethe affinity of the bioactive peptide. Furthermore, it may be desirableto put the identified random region of the bioactive peptide into otherpresentation structures, or to alter the sequence of the constant regionof the presentation structure, to alter the conformation/shape of thebioactive peptide. It may also be desirable to “walk” around a potentialbinding site, in a manner similar to the mutagenesis of a bindingpocket, by keeping one end of the ligand region constant and randomizingthe other end to shift the binding of the peptide around.

In a preferred embodiment, either the bioactive peptide or the bioactivenucleic acid encoding it is used to identify target molecules, i.e. themolecules with which the bioactive peptide interacts. As will beappreciated by those in the art, there may be primary target molecules,to which the bioactive peptide binds or acts upon directly, and theremay be secondary target molecules, which are part of the signallingpathway affected by the bioactive peptide; these might be termed“validated targets”.

In a preferred embodiment, the bioactive peptide is a drug. As will beappreciated by those in the art, the structure of the cyclic peptide maybe modeled and used in rational drug design to synthesize agents thatmimic the interaction of the cyclic peptide with its' target. Drugs mayalso be modeled based on the three dimensional structure of the peptidebound to its target. Drugs so modeled may have structures that aresimilar to or unrelated to the starting structure of the cyclic peptideor the cyclic peptide bound to its target. Alternatively, highthroughput screens can be used to identify small molecules capable ofcompeting with the cyclic peptide for its target.

In a preferred embodiment, the bioactive cyclic peptide may be used asthe starting point for designing/synthesizing derivative molecules withsimilar or more favorable properties for use as a drug. For example,individual amino acids, specific chemical groups, etc., can be replacedand the derivative molecule tested for use as a drug. Both naturallyoccurring and synthetic amino acid analogs (see below for definition)can be introduced in to the derivative molecule to optimize propertiessuch as binding, stability, pharmocokinectics. Preferably, thederivative molecule has one or more of the following properties:improved stability, higher binding affinity, improved specificity forthe target, improved pharmocokinetics, i.e., absorption, distribution,resistance to degradation, etc.

In a preferred embodiment, the bioactive peptide is used to pull outtarget molecules. For example, as outlined herein, if the targetmolecules are proteins, the use of epitope tags, purification sequences,or affinity tags can allow the purification of primary target moleculesvia biochemical means (co-immunoprecipitation, affinity columns, etc.).Alternatively, the peptide, when expressed in bacteria and purified, canbe used as a probe against a bacterial cDNA expression library made frommRNA of the target cell type. Or, peptides can be used as “bait” ineither yeast or mammalian two or three hybrid systems. Such interactioncloning approaches have been very useful to isolate DNA-binding proteinsand other interacting protein components. The peptide(s) can be combinedwith other pharmacologic activators to study the epistatic relationshipsof signal transduction pathways in question. It is also possible tosynthetically prepare labeled peptide and use it to screen a cDNAlibrary expressed in bacteriophage for those cDNAs which bind thepeptide. Furthermore, it is also possible that one could use cDNAcloning via retroviral libraries to “complement” the effect induced bythe peptide. In such a strategy, the peptide would be required to bestoichiometrically titrating away some important factor for a specificsignaling pathway. If this molecule or activity is replenished byover-expression of a cDNA from within a cDNA library, then one can clonethe target. Similarly, cDNAs cloned by any of the above yeast orbacteriophage systems can be reintroduced to mammalian cells in thismanner to confirm that they act to complement function in the system thepeptide acts upon.

In a preferred embodiment, target molecules are identified byincorporating an affinity tagged amino acid residue into the sequence ofthe cyclic peptide. For example, incorporation of a cysteine allows forthe chemical conjugation of the cyclic peptide to a solid support matrixvia a disulfide bond. In particular, target molecules that bind tofunctional cyclic peptides are isolated and identified using affinitytagged amino acids.

In a preferred embodiment, the cysteine contributed by the extein isuniquely alkylated with an affinity reagent as part of the synthesis ofthe peptide to allow affinity extraction and identification of targetmolecules using HPLC-mass spectrometry methods. Cysteine-alkylatedcyclic peptide analogs are tested for function, and if functional,target molecules are affinity extracted using methods well known in theart. If the cysteine-alkylated peptide analogs are not functional,synthetic cyclic peptide analogs are constructed with cysteine-affinitytag amino acid analogs in other positions and tested for function. Inalternative embodiments, lysine affinity tagged amino acids are used.

Alternatively, if an affinity tagged amino acid cannot be produced invivo, the tag can be introduced in vitro and tested in vivo forfunction.

Any amino acid which can be used as a affinity tag may be used in themethods of the invention. This includes both naturally occurring andsynthetic amino acid analogs which can be introduced into the cyclicpeptide to facilitate chemical conjugation or binding to a solid supportmatrix. Thus “amino acid”, or “peptide residue”, as used herein meansboth naturally occurring and synthetic amino acids. For example,homo-phenylalanine, citrulline, and norleucine are considered aminoacids for the purposes of the invention. “Amino acid” also includesimino acid residues such as proline and hydroxyproline. In addition, anyamino acid can be replaced by the same amino acid but of the oppositechirality. Thus, any amino acid naturally occurring in theL-configuration (which may also be referred to as the R or S, dependingupon the structure of the chemical entity) may be replaced with an aminoacid of the same chemical structural type, but of the oppositechirality, generally referred to as the D-amino acid but which canadditionally be referred to as the R- or the S-, depending upon itscomposition and chemical configuration. Such derivatives have theproperty of greatly increased stability, and therefore are advantageousin the formulation of compounds which may have longer in vivo halflives, when administered by oral, intravenous, intramuscular,intraperitoneal, topical, rectal, intraocular, or other routes.

In the preferred embodiment, the amino acids are in the (S) orL-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradations. Proteins including non-naturally occurringamino acids may be synthesized or in some cases, made recombinantly; seevan Hest et al., FEBS Lett 428:(1-2) 68-70 May 22, 1998 and Tang et al.,Abstr. Pap Am. Chem. S218:U138-U138 Part 2 Aug. 22, 1999, both of whichare expressly incorporated by reference herein.

Aromatic amino acids may be replaced with D- or L-naphylalanine, D- orL-Phenylglycine, D- or L-2-thieneylalanine, D- or L-1-, 2-, 3- or4-pyreneylalanine, D- or L-3-thieneylalanine, D- orL-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- orL-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine,D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine,D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- orL-p-methoxybiphenylphenylalanine, D- or L-2-indole(alkyl)alanines, andD- or L-alkylainines where alkyl may be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, non-acidic amino acids, of C1-C20.

Acidic amino acids can be substituted with non-carboxylate amino acidswhile maintaining a negative charge, and derivatives or analogs thereof,such as the non-limiting examples of (phosphono)alanine, glycine,leucine, isoleucine, threonine, or serine; or sulfated (e.g.,—SO.sub.3H) threonine, serine, tyrosine.

Other substitutions may include unnatural hyroxylated amino acids maymade by combining “alkyl” with any natural amino acid. The term “alkyl”as used herein refers to a branched or unbranched saturated hydrocarbongroup of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl,isoptopyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl,hexadecyl, eicosyl, tetracisyl and the like. Alkyl includes heteroalkyl,with atoms of nitrogen, oxygen and sulfur. Preferred alkyl groups hereincontain 1 to 12 carbon atoms. Basic amino acids may be substituted withalkyl groups at any position of the naturally occurring amino acidslysine, arginine, ornithine, citrulline, or (guanidino)-acetic acid, orother (guanidino)alkyl-acetic acids, where “alkyl” is define as above.Nitrile derivatives (e.g., containing the CN-moiety in place of COOH)may also be substituted for asparagine or glutamine, and methioninesulfoxide may be substituted for methionine. Methods of preparation ofsuch peptide derivatives are well known to one skilled in the art.

In addition, any amide linkage can be replaced by a ketomethylenemoiety. Such derivatives are expected to have the property of increasedstability to degradation by enzymes, and therefore possess advantagesfor the formulation of compounds which may have increased in vivo halflives, as administered by oral, intravenous, intramuscular,intraperitoneal, topical, rectal, intraocular, or other routes.

Additional amino acid modifications of amino acids of to the presentinvention may include the following: Cysteinyl residues may be reactedwith alpha-haloacetates (and corresponding amines), such as2-chloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues may also bederivatized by reaction with compounds such as bromotrifluoroacetone,alpha-bromo-beta-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues may be derivatized by reaction with compounds such asdiethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain, and para-bromophenacyl bromide mayalso be used; e.g., where the reaction is preferably performed in 0.1 Msodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds suchas succinic or other carboxylic acid anhydrides. Derivatization withthese agents is expected to have the effect of reversing the charge ofthe lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include compounds such asimidoesters/e.g., as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin according to known method steps.Derivatization of arginine residues requires that the reaction beperformed in alkaline conditions because of the high pKa of theguanidine functional group. Furthermore, these reagents may react withthe groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se is well-known, suchas for introducing spectral labels into tyrosyl residues by reactionwith aromatic diazonium compounds or tetranitromethane. N-acetylimidizoland tetranitromethane may be used to form O-acetyl tyrosyl species and3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R′-N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermoreaspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of the present invention.

Examples of affinity labeled amino acids useful for extraction of targetmolecules include lysine-epsilon amino biotin, or lysine reacted withamine-specific biotinylation reagents such as biotin-NHS ester andsulfo-NHS biotin.

Spacers may be incorporated between the affinity element and the peptideto relieve steric restraints between the affinity tag and a cyclicpeptide bound to a target molecule. A spacer which may be used withaffinity tagged lysine is NHS-LC-biotin (Pierce Chemical CO., RockfordIll.), although other spacers as are known in the art also may be used.

Examples of spacers which can be used with affinity tagged cysteinesinclude cysteine reacted with iodoacetamido-biotin,biotin-hexyl-3′-(2′-pyridyldithio) propionamide (a 29 Å spacer fromPierce Chemical), iodoacetyl-LC-biotin (27 Å spacer) or biotin-BMCC witha 32 Å spacer (Pierce Chemical). An example of a spacer used withaffinity tagged cysteine is shown in Structure 1:

Alternatively, as part of the solid phase synthesis of the peptide,affinity tags may be synthesized branching off from the cysteine orlysine. In this case, the spacer consists of a defined number (i.e. n)of amino acids branching off the side chain of the cysteine or lysine oranother residue of the cyclic peptide. Preferably, n=1 to 40. Thisallows for spacers of variable length, ranging from 3 Å to 100 Å ormore. Gycines, because of their flexibility, are preferred because asterically bulky target molecules bound to the cyclic peptide can beaccommodated. The affinity tag is inserted at the end of the side chainas illustrated in Structure 2:

In a preferred embodiment, the spacer is at least one protein diameterlong (20-40 Å). When the interacting target molecule is part of a largecomplex, the spacer is up to at least two protein diameters (40-80 Å).

Once primary target molecules have been identified, secondary targetmolecules may be identified in the same manner, using the primary targetas the “bait”. In this manner, signaling pathways may be elucidated.Similarly, bioactive peptides specific for secondary target moleculesmay also be discovered, to allow a number of bioactive peptides to acton a single pathway, for example for combination therapies.

The screening methods of the present invention may be useful to screen alarge number of cell types under a wide variety of conditions.Generally, the host cells are cells that are involved in disease states,and they are tested or screened under conditions that normally result inundesirable consequences on the cells. When a suitable bioactive peptideis found, the undesirable effect may be reduced or eliminated.Alternatively, normally desirable consequences may be reduced oreliminated, with an eye towards elucidating the cellular mechanismsassociated with the disease state or signalling pathway.

In a preferred embodiment, the present methods are useful in cancerapplications. The ability to rapidly and specifically kill tumor cellsis a cornerstone of cancer chemotherapy. In general, using the methodsof the present invention, random libraries can be introduced into anytumor cell (primary or cultured), and peptides identified which bythemselves induce apoptosis, cell death, loss of cell division ordecreased cell growth. This may be done de novo, or by biasedrandomization toward known peptide agents, such as angiostatin, whichinhibits blood vessel wall growth. Alternatively, the methods of thepresent invention can be combined with other cancer therapeutics (e.g.drugs or radiation) to sensitize the cells and thus induce rapid andspecific apoptosis, cell death, loss of cell division or decreased cellgrowth after exposure to a secondary agent. Similarly, the presentmethods may be used in conjunction with known cancer therapeutics toscreen for agonists to make the therapeutic more effective or lesstoxic. This is particularly preferred when the chemotherapeutic is veryexpensive to produce such as taxol.

Known oncogenes such as v-Abl, v-Src, v-Ras, and others, induce atransformed phenotype leading to abnormal cell growth when transfectedinto certain cells. This is also a major problem with micro-metastases.Thus, in a preferred embodiment, non-transformed cells can betransfected with these oncogenes, and then random libraries introducedinto these cells, to select for bioactive peptides which reverse orcorrect the transformed state. One of the signal features of oncogenetransformation of cells is the loss of contact inhibition and theability to grow in soft-agar. When transforming viruses are constructedcontaining v-Abl, v-Src, or v-Ras in IRES-puro retroviral vectors,infected into target 3T3 cells, and subjected to puromycin selection,all of the 3T3 cells hyper-transform and detach from the plate. Thecells may be removed by washing with fresh medium. This can serve as thebasis of a screen, since cells which express a bioactive peptide willremain attached to the plate and form colonies.

Similarly, the growth and/or spread of certain tumor types is enhancedby stimulatory responses from growth factors and cytokines (PDGF, EGF,Heregulin, and others) which bind to receptors on the surfaces ofspecific tumors. In a preferred embodiment, the methods of the inventionare used to inhibit or stop tumor growth and/or spread, by findingbioactive peptides capable of blocking the ability of the growth factoror cytokine to stimulate the tumor cell. The introduction of randomlibraries into specific tumor cells with the addition of the growthfactor or cytokine, followed by selection of bioactive peptides whichblock the binding, signaling, phenotypic and/or functional responses ofthese tumor cells to the growth factor or cytokine in question.

Similarly, the spread of cancer cells (invasion and metastasis) is asignificant problem limiting the success of cancer therapies. Theability to inhibit the invasion and/or migration of specific tumor cellswould be a significant advance in the therapy of cancer. Tumor cellsknown to have a high metastatic potential (for example, melanoma, lungcell carcinoma, breast and ovarian carcinoma) can have random librariesintroduced into them, and peptides selected which in a migration orinvasion assay, inhibit the migration and/or invasion of specific tumorcells. Particular applications for inhibition of the metastaticphenotype, which could allow a more specific inhibition of metastasis,include the metastasis suppressor gene NM23, which codes for adinucleoside diphosphate kinase. Thus intracellular peptide activatorsof this gene could block metastasis, and a screen for its upregulation(by fusing it to a reporter gene) would be of interest. Many oncogenesalso enhance metastasis. Peptides which inactivate or counteract mutatedRAS oncogenes, v-MOS, v-RAF, A-RAF, v-SRC, v-FES, and v-FMS would alsoact as anti-metastatics. Peptides which act intracellularly to block therelease of combinations of proteases required for invasion, such as thematrix metalloproteases and urokinase, could also be effectiveantimetastatics.

In a preferred embodiment, the random libraries of the present inventionare introduced into tumor cells known to have inactivated tumorsuppressor genes, and successful reversal by either reactivation orcompensation of the knockout would be screened by restoration of thenormal phenotype. A major example is the reversal of p53-inactivatingmutations, which are present in 50% or more of all cancers. Since p53'sactions are complex and involve its action as a transcription factor,there are probably numerous potential ways a peptide or small moleculederived from a peptide could reverse the mutation. One example would beupregulation of the immediately downstream cyclin-dependent kinasep21CIP1/WAF1. To be useful such reversal would have to work for many ofthe different known p53 mutations. This is currently being approached bygene therapy; one or more small molecules which do this might bepreferable.

Another example involves screening of bioactive peptides which restorethe constitutive function of the brca-1 or brca-2 genes, and other tumorsuppressor genes important in breast cancer such as the adenomatouspolyposis coli gene (APC) and the Drosophila discs-large gene (Dig),which are components of cell-cell junctions. Mutations of brca-1 areimportant in hereditary ovarian and breast cancers, and constitute anadditional application of the present invention.

In a preferred embodiment, the methods of the present invention are usedto create novel cell lines from cancers from patients. A retrovirallydelivered short peptide which inhibits the final common pathway ofprogrammed cell death should allow for short- and possibly long-termcell lines to be established. Conditions of in vitro culture andinfection of human leukemia cells will be established. There is a realneed for methods which allow the maintenance of certain tumor cells inculture long enough to allow for physiological and pharmacologicalstudies. Currently, some human cell lines have been established by theuse of transforming agents such as Epstein-Barr virus that considerablyalters the existing physiology of the cell. On occasion, cells will growon their own in culture but this is a random event. Programmed celldeath (apoptosis) occurs via complex signaling pathways within cellsthat ultimately activate a final common pathway producing characteristicchanges in the cell leading to a non-inflammatory destruction of thecell. It is well known that tumor cells have a high apoptotic index, orpropensity to enter apoptosis in vivo. When cells are placed in culture,the in vivo stimuli for malignant cell growth are removed and cellsreadily undergo apoptosis. The objective would be to develop thetechnology to establish cell lines from any number of primary tumorcells, for example primary human leukemia cells, in a reproduciblemanner without altering the native configuration of the signalingpathways in these cells. By introducing nucleic acids encoding peptideswhich inhibit apoptosis, increased cell survival in vitro, and hence theopportunity to study signalling transduction pathways in primary humantumor cells, is accomplished. In addition, these methods may be used forculturing primary cells, i.e. non-tumor cells.

In a preferred embodiment, the present methods are useful incardiovascular applications. In a preferred embodiment, cardiomyocytesmay be screened for the prevention of cell damage or death in thepresence of normally injurious conditions, including, but not limitedto, the presence of toxic drugs (particularly chemotherapeutic drugs),for example, to prevent heart failure following treatment withadriamycin; anoxia, for example in the setting of coronary arteryocclusion; and autoimmune cellular damage by attack from activatedlymphoid cells (for example as seen in post viral myocarditis andlupus). Candidate bioactive peptides are inserted into cardiomyocytes,the cells are subjected to the insult, and bioactive peptides areselected that prevent any or all of: apoptosis; membrane depolarization(i.e. decrease arrythmogenic potential of insult); cell swelling; orleakage of specific intracellular ions, second messengers and activatingmolecules (for example, arachidonic acid and/or lysophosphatidic acid).

In a preferred embodiment, the present methods are used to screen fordiminished arrhythmia potential in cardiomyocytes. The screens comprisethe introduction of the candidate nucleic acids encoding candidatebioactive peptides, followed by the application of arrythmogenicinsults, with screening for bioactive peptides that block specificdepolarization of cell membrane. This may be detected using patchclamps, or via fluorescence techniques). Similarly, channel activity(for example, potassium and chloride channels) in cardiomyocytes couldbe regulated using the present methods in order to enhance contractilityand prevent or diminish arrhythmias.

In a preferred embodiment, the present methods are used to screen forenhanced contractile properties of cardiomyocytes and diminish heartfailure potential. The introduction of the libraries of the inventionfollowed by measuring the rate of change of myosinpolymerization/depolymerization using fluorescent techniques can bedone. Bioactive peptides which increase the rate of change of thisphenomenon can result in a greater contractile response of the entiremyocardium, similar to the effect seen with digitalis.

In a preferred embodiment, the present methods are useful to identifyagents that will regulate the intracellular and sarcolemmal calciumcycling in cardiomyocytes in order to prevent arrhythmias. Bioactivepeptides are selected that regulate sodium-calcium exchange, sodiumproton pump function, and regulation of calcium-ATPase activity.

In a preferred embodiment, the present methods are useful to identifyagents that diminish embolic phenomena in arteries and arteriolesleading to strokes (and other occlusive events leading to kidney failureand limb ischemia) and angina precipitating a myocardial infarct areselected. For example, bioactve peptides which will diminish theadhesion of platelets and leukocytes, and thus diminish the occlusionevents. Adhesion in this setting can be inhibited by the libraries ofthe invention being inserted into endothelial cells (quiescent cells, oractivated by cytokines, i.e. IL-1, and growth factors, i.e. PDGF/EGF)and then screening for peptides that either: 1) down regulate adhesionmolecule expression on the surface of the endothelial cells (bindingassay); 2) block adhesion molecule activation on the surface of thesecells (signaling assay); or 3) release in an autocrine manner peptidesthat block receptor binding to the cognate receptor on the adheringcell.

Embolic phenomena can also be addressed by activating proteolyticenzymes on the cell surfaces of endothelial cells, and thus releasingactive enzyme which can digest blood clots. Thus, delivery of thelibraries of the invention to endothelial cells is done, followed bystandard fluorogenic assays, which will allow monitoring of proteolyticactivity on the cell surface towards a known substrate. Bioactivepeptides can then be selected which activate specific enzymes towardsspecific substrates.

In a preferred embodiment, arterial inflammation in the setting ofvasculitis and post-infarction can be regulated by decreasing thechemotactic responses of leukocytes and mononuclear leukocytes. This canbe accomplished by blocking chemotactic receptors and their respondingpathways on these cells. Candidate bioactve libraries can be insertedinto these cells, and the chemotactic response to diverse chemokines(for example, to the IL-8 family of chemokines, RANTES) inhibited incell migration assays.

In a preferred embodiment, arterial restenosis following coronaryangioplasty can be controlled by regulating the proliferation ofvascular intimal cells and capillary and/or arterial endothelial cells.Candidate bioactve peptide libraries can be inserted into these celltypes and their proliferation in response to specific stimuli monitored.One application may be intracellular peptides which block the expressionor function of c-myc and other oncogenes in smooth muscle cells to stoptheir proliferation. A second application may involve the expression oflibraries in vascular smooth muscle cells to selectively induce theirapoptosis. Application of small molecules derived from these peptidesmay require targeted drug delivery; this is available with stents,hydrogel coatings, and infusion-based catheter systems. Peptides whichdownregulate endothelin-1A receptors or which block the release of thepotent vasoconstrictor and vascular smooth muscle cell mitogenendothelin-1 may also be candidates for therapeutics. Peptides can beisolated from these libraries which inhibit growth of these cells, orwhich prevent the adhesion of other cells in the circulation known torelease autocrine growth factors, such as platelets (PDGF) andmononuclear leukocytes.

The control of capillary and blood vessel growth is an important goal inorder to promote increased blood flow to ischemic areas (growth), or tocut-off the blood supply (angiogenesis inhibition) of tumors. Candidatebioactive peptide libraries can be inserted into capillary endothelialcells and their growth monitored. Stimuli such as low oxygen tension andvarying degrees of angiogenic factors can regulate the responses, andpeptides isolated that produce the appropriate phenotype. Screening forantagonism of vascular endothelial cell growth factor, important inangiogenesis, would also be useful.

In a preferred embodiment, the present methods are useful in screeningfor decreases in atherosclerosis producing mechanisms to find peptidesthat regulate LDL and HDL metabolism. Candidate libraries can beinserted into the appropriate cells (including hepatocytes, mononuclearleukocytes, endothelial cells) and peptides selected which lead to adecreased release of LDL or diminished synthesis of LDL, or converselyto an increased release of HDL or enhanced synthesis of HDL. Bioactivepeptides can also be isolated from candidate libraries which decreasethe production of oxidized LDL, which has been implicated inatherosclerosis and isolated from atherosclerotic lesions. This couldoccur by decreasing its expression, activating reducing systems orenzymes, or blocking the activity or production of enzymes implicated inproduction of oxidized LDL, such as 15-lipoxygenase in macrophages.

In a preferred embodiment, the present methods are used in screens toregulate obesity via the control of food intake mechanisms ordiminishing the responses of receptor signaling pathways that regulatemetabolism. Bioactive peptides that regulate or inhibit the responses ofneuropeptide Y (NPY), cholecystokinin and galanin receptors, areparticularly desirable. Candidate libraries can be inserted into cellsthat have these receptors cloned into them, and inhibitory peptidesselected that are secreted in an autocrine manner that block thesignaling responses to galanin and NPY. In a similar manner, peptidescan be found that regulate the leptin receptor.

In a preferred embodiment, the present methods are useful inneurobiology applications. Candidate libraries may be used for screeningfor anti-apoptotics for preservation of neuronal function and preventionof neuronal death. Initial screens would be done in cell culture. Oneapplication would include prevention of neuronal death, by apoptosis, incerebral ischemia resulting from stroke. Apoptosis is known to beblocked by neuronal apoptosis inhibitory protein (NAIP); screens for itsupregulation, or effecting any coupled step could yield peptides whichselectively block neuronal apoptosis. Other applications includeneurodegenerative diseases such as Alzheimer's disease and Huntington'sdisease.

In a preferred embodiment, the present methods are useful in bonebiology applications. Osteoclasts are known to play a key role in boneremodeling by breaking down “old” bone, so that osteoblasts can lay down“new” bone. In osteoporosis one has an imbalance of this process.Osteoclast overactivity can be regulated by inserting candidatelibraries into these cells, and then looking for bioactve peptides thatproduce: 1) a diminished processing of collagen by these cells; 2)decreased pit formation on bone chips; and 3) decreased release ofcalcium from bone fragments.

The present methods may also be used to screen for agonists of bonemorphogenic proteins, hormone mimetics to stimulate, regulate, orenhance new bone formation (in a manner similar to parathyroid hormoneand calcitonin, for example). These have use in osteoporosis, for poorlyhealing fractures, and to accelerate the rate of healing of newfractures. Furthermore, cell lines of connective tissue origin can betreated with candidate libraries and screened for their growth,proliferation, collagen stimulating activity, and/or prolineincorporating ability on the target osteoblasts. Alternatively,candidate libraries can be expressed directly in osteoblasts orchondrocytes and screened for increased production of collagen or bone.

In a preferred embodiment, the present methods are useful in skinbiology applications. Keratinocyte responses to a variety of stimuli mayresult in psoriasis, a proliferative change in these cells. Candidatelibraries can be inserted into cells removed from active psoriaticplaques, and bioactive peptides isolated which decrease the rate ofgrowth of these cells.

In a preferred embodiment, the present methods are useful in theregulation or inhibition of keloid formation (i.e. excessive scarring).Candidate libraries inserted into skin connective tissue cells isolatedfrom individuals with this condition, and bioactive peptides isolatedthat decrease proliferation, collagen formation, or prolineincorporation. Results from this work can be extended to treat theexcessive scarring that also occurs in burn patients. If a commonpeptide motif is found in the context of the keloid work, then it can beused widely in a topical manner to diminish scarring post burn.

Similarly, wound healing for diabetic ulcers and other chronic “failureto heal” conditions in the skin and extremities can be regulated byproviding additional growth signals to cells which populate the skin anddermal layers. Growth factor mimetics may in fact be very useful forthis condition. Candidate libraries can be inserted into skin connectivetissue cells, and bioactive peptides isolated which promote the growthof these cells under “harsh” conditions, such as low oxygen tension, lowpH, and the presence of inflammatory mediators.

Cosmeceutical applications of the present invention include the controlof melanin production in skin melanocytes. A naturally occurringpeptide, arbutin, is a tyrosine hydroxylase inhibitor, a key enzyme inthe synthesis of melanin. Candidate libraries can be inserted intomelanocytes and known stimuli that increase the synthesis of melaninapplied to the cells. Bioactive peptides can be isolated that inhibitthe synthesis of melanin under these conditions.

In a preferred embodiment, the present methods are useful inendocrinology applications. The retroviral peptide library technologycan be applied broadly to any endocrine, growth factor, cytokine orchemokine network which involves a signaling peptide or protein thatacts in either an endocrine, paracrine or autocrine manner that binds ordimerizes a receptor and activates a signaling cascade that results in aknown phenotypic or functional outcome. The methods are applied so as toisolate a peptide which either mimics the desired hormone (i.e.,insulin, leptin, calcitonin, PDGF, EGF, EPO, GMCSF, IL1-17, mimetics) orinhibits its action by either blocking the release of the hormone,blocking its binding to a specific receptor or carrier protein (forexample, CRF binding protein), or inhibiting the intracellular responsesof the specific target cells to that hormone. Selection of peptideswhich increase the expression or release of hormones from the cellswhich normally produce them could have broad applications to conditionsof hormonal deficiency.

In a preferred embodiment, the present methods are useful in infectiousdisease applications. Viral latency (herpes viruses such as CMV, EBV,HBV, and other viruses such as HIV) and their reactivation are asignificant problem, particularly in immunosuppressed patients (patientswith AIDS and transplant patients). The ability to block thereactivation and spread of these viruses is an important goal. Celllines known to harbor or be susceptible to latent viral infection can beinfected with the specific virus, and then stimuli applied to thesecells which have been shown to lead to reactivation and viralreplication. This can be followed by measuring viral titers in themedium and scoring cells for phenotypic changes. Candidate libraries canthen be inserted into these cells under the above conditions, andpeptides isolated which block or diminish the growth and/or release ofthe virus. As with chemotherapeutics, these experiments can also be donewith drugs which are only partially effective towards this outcome, andbioactive peptides isolated which enhance the virucidal effect of thesedrugs. Bioactive peptides may also be tested for the ability to blocksome aspect of viral assembly, viral replication, entry or infectiouscycle.

One example of many is the ability to block HIV-1 infection. HIV-1requires CD4 and a co-receptor which can be one of several seventransmembrane G-protein coupled receptors. In the case of the infectionof macrophages, CCR-5 is the required co-receptor, and there is strongevidence that a block on CCR-5 will result in resistance to HIV-1infection. There are two lines of evidence for this statement First, itis known that the natural ligands for CCR-5, the CC chemokines RANTES,MIP1a and MIP1b are responsible for CD8+ mediated resistance to HIV.Second, individuals homozygous for a mutant allele of CCR-5 arecompletely resistant to HIV infection. Thus, an inhibitor of theCCR-5/HIV interaction would be of enormous interest to both biologistsand clinicians. The extracellular anchored constructs offer superb toolsfor such a discovery. Into the transmembrane, epitope tagged,glycine-serine tethered constructs (ssTM V G20 E TM), one can place arandom, cyclized peptide library of the general sequence CNNNNNNNNNNC orC—(X)_(n)—C. Then one infects a cell line that expresses CCR-5 withretroviruses containing this library. Using an antibody to CCR-5 one canuse FACS to sort desired cells based on the binding of this antibody tothe receptor. All cells which do not bind the antibody will be assumedcontain inhibitors of this antibody binding site. These inhibitors, inthe retroviral construct can be further assayed for their ability toinhibit HIV-1 entry.

Viruses are known to enter cells using specific receptors to bind tocells (for example, HIV uses CD4, coronavirus uses CD13, murine leukemiavirus uses transport protein, and measles virus uses CD44) and to fusewith cells (HIV uses chemokine receptor). Candidate libraries can beinserted into target cells known to be permissive to these viruses, andbioactive peptides isolated which block the ability of these viruses tobind and fuse with specific target cells.

Intein libraries may also be used to screen for cyclic peptides whichblock HIV-1 infection. For example, inteins can be designed such thatcyclized peptides are secreted from cells where they can bind to CCR5and antagonize HIV-1 binding.

In a preferred embodiment, the present invention finds use withinfectious organisms. Intracellular organisms such as mycobacteria,listeria, salmonella, pneumocystis, yersinia, leishmania, T. cruzi, canpersist and replicate within cells, and become active inimmunosuppressed patients. There are currently drugs on the market andin development which are either only partially effective or ineffectiveagainst these organisms. Candidate libraries can be inserted intospecific cells infected with these organisms (pre- or post-infection),and bioactive peptides selected which promote the intracellulardestruction of these organisms in a manner analogous to intracellular“antibiotic peptides” similar to magainins. In addition peptides can beselected which enhance the cidal properties of drugs already underinvestigation which have insufficient potency by themselves, but whencombined with a specific peptide from a candidate library, aredramatically more potent through a synergistic mechanism. Finally,bioactive peptides can be isolated which alter the metabolism of theseintracellular organisms, in such a way as to terminate theirintracellular life cycle by inhibiting a key organismal event.

Antibiotic drugs that are widely used have certain dose dependent,tissue specific toxicities. For example renal toxicity is seen with theuse of gentamicin, tobramycin, and amphotericin; hepatotoxicity is seenwith the use of INH and rifampin; bone marrow toxicity is seen withchloramphenicol; and platelet toxicity is seen with ticarcillin, etc.These toxicities limit their use. Candidate libraries can be introducedinto the specific cell types where specific changes leading to cellulardamage or apoptosis by the antibiotics are produced, and bioactivepeptides can be isolated that confer protection, when these cells aretreated with these specific antibiotics.

Furthermore, the present invention finds use in screening for bioactivepeptides that block antibiotic transport mechanisms. The rapid secretionfrom the blood stream of certain antibiotics limits their usefulness.For example penicillins are rapidly secreted by certain transportmechanisms in the kidney and choroid plexus in the brain. Probenecid isknown to block this transport and increase serum and tissue levels.Candidate agents can be inserted into specific cells derived from kidneycells and cells of the choroid plexus known to have active transportmechanisms for antibiotics. Bioactive peptides can then be isolatedwhich block the active transport of specific antibiotics and thus extendthe serum half-life of these drugs.

In a preferred embodiment, the present methods are useful in drugtoxicities and drug resistance applications. Drug toxicity is asignificant clinical problem. This may manifest itself as specifictissue or cell damage with the result that the drug's effectiveness islimited. Examples include myeloablation in high dose cancerchemotherapy, damage to epithelial cells lining the airway and gut, andhair loss. Specific examples include adriamycin induced cardiomyocytedeath, cisplatinin-induced kidney toxicity, vincristine-induced gutmobility disorders, and cyclosporin-induced kidney damage. Candidatelibraries can be introduced into specific cell types with characteristicdrug-induced phenotypic or functional responses, in the presence of thedrugs, and agents isolated which reverse or protect the specific celltype against the toxic changes when exposed to the drug. These effectsmay manifest as blocking the drug induced apoptosis of the cell ofinterest, thus initial screens will be for survival of the cells in thepresence of high levels of drugs or combinations of drugs used incombination chemotherapy.

Drug toxicity may be due to a specific metabolite produced in the liveror kidney which is highly toxic to specific cells, or due to druginteractions in the liver which block or enhance the metabolism of anadministered drug. Candidate libraries can be introduced into liver orkidney cells following the exposure of these cells to the drug known toproduce the toxic metabolite. Bioactive peptides can be isolated whichalter how the liver or kidney cells metabolize the drug, and specificagents identified which prevent the generation of a specific toxicmetabolite. The generation of the metabolite can be followed by massspectrometry, and phenotypic changes can be assessed by microscopy. Sucha screen can also be done in cultured hepatocytes, cocultured withreadout cells which are specifically sensitive to the toxic metabolite.Applications include reversible (to limit toxicity) inhibitors ofenzymes involved in drug metabolism.

Multiple drug resistance, and hence tumor cell selection, outgrowth, andrelapse, leads to morbidity and mortality in cancer patents. Candidatelibraries can be introduced into tumor cell lines (primary and cultured)that have demonstrated specific or multiple drug resistance. Bioactivepeptides can then be identified which confer drug sensitivity when thecells are exposed to the drug of interest, or to drugs used incombination chemotherapy. The readout can be the onset of apoptosis inthese cells, membrane permeability changes, the release of intracellularions and fluorescent markers. The cells in which multidrug resistanceinvolves membrane transporters can be preloaded with fluorescenttransporter substrates, and selection carried out for peptides whichblock the normal efflux of fluorescent drug from these cells. Candidatelibraries are particularly suited to screening for peptides whichreverse poorly characterized or recently discovered intracellularmechanisms of resistance or mechanisms for which few or nochemosensitizers currently exist, such as mechanisms involving LRP (lungresistance protein). This protein has been implicated in multidrugresistance in ovarian carcinoma, metastatic malignant melanoma, andacute myeloid leukemia. Particularly interesting examples includescreening for agents which reverse more than one important resistancemechanism in a single cell, which occurs in a subset of the most drugresistant cells, which are also important targets. Applications wouldinclude screening for peptide inhibitors of both MRP (multidrugresistance related protein) and LRP for treatment of resistant cells inmetastatic melanoma, for inhibitors of both p-glycoprotein and LRP inacute myeloid leukemia, and for inhibition (by any mechanism) of allthree proteins for treating pan-resistant cells.

In a preferred embodiment, the present methods are useful in improvingthe performance of existing or developmental drugs. First passmetabolism of orally administered drugs limits their oralbioavailability, and can result in diminished efficacy as well as theneed to administer more drug for a desired effect. Reversible inhibitorsof enzymes involved in first pass metabolism may thus be a usefuladjunct enhancing the efficacy of these drugs. First pass metabolismoccurs in the liver, thus inhibitors of the corresponding catabolicenzymes may enhance the effect of the cognate drugs. Reversibleinhibitors would be delivered at the same time as, or slightly before,the drug of interest. Screening of candidate libraries in hepatocytesfor inhibitors (by any mechanism, such as protein downregulation as wellas a direct inhibition of activity) of particularly problematicalisozymes would be of interest. These include the CYP3A4 isozymes ofcytochrome P450, which are involved in the first pass metabolism of theanti-HIV drugs saquinavir and indinavir. Other applications couldinclude reversible inhibitors of UDP-glucuronyltransferases,sulfotransferases, N-acetyltransferases, epoxide hydrolases, andglutathione S-transferases, depending on the drug. Screens would be donein cultured hepatocytes or liver microsomes, and could involveantibodies recognizing the specific modification performed in the liver,or co-cultured readout cells, if the metabolite had a differentbioactivity than the untransformed drug. The enzymes modifying the drugwould not necessarily have to be known, if screening was for lack ofalteration of the drug.

In a preferred embodiment, the present methods are useful inimmunobiology, inflammation, and allergic response applications.Selective regulation of T lymphocyte responses is a desired goal inorder to modulate immune-mediated diseases in a specific manner.Candidate libraries can be introduced into specific T cell subsets (TH1,TH2, CD4+, CD8+, and others) and the responses which characterize thosesubsets (cytokine generation, cytotoxicity, proliferation in response toantigen being presented by a mononuclear leukocyte, and others) modifiedby members of the library. Agents can be selected which increase ordiminish the known T cell subset physiologic response. This approachwill be useful in any number of conditions, including: 1) autoimmunediseases where one wants to induce a tolerant state (select a peptidethat inhibits T cell subset from recognizing a self-antigen bearingcell); 2) allergic diseases where one wants to decrease the stimulationof IgE producing cells (select peptide which blocks release from T cellsubsets of specific B-cell stimulating cytokines which induce switch toIgE production); 3) in transplant patients where one wants to induceselective immunosuppression (select peptide that diminishesproliferative responses of host T cells to foreign antigens); 4) inlymphoproliferative states where one wants to inhibit the growth orsensitize a specific T cell tumor to chemotherapy and/or radiation; 5)in tumor surveillance where one wants to inhibit the killing ofcytotoxic T cells by Fas ligand bearing tumor cells; and 5) in T cellmediated inflammatory diseases such as Rheumatoid arthritis, Connectivetissue diseases (SLE), Multiple sclerosis, and inflammatory boweldisease, where one wants to inhibit the proliferation of disease-causingT cells (promote their selective apoptosis) and the resulting selectivedestruction of target tissues (cartilage, connective tissue,oligodendrocytes, gut endothelial cells, respectively).

Regulation of B cell responses will permit a more selective modulationof the type and amount of immunoglobulin made and secreted by specific Bcell subsets. Candidate libraries can be inserted into B cells andbioactive peptides selected which inhibit the release and synthesis of aspecific immunoglobulin. This may be useful in autoimmune diseasescharacterized by the overproduction of auto antibodies and theproduction of allergy causing antibodies, such as IgE. Agents can alsobe identified which inhibit or enhance the binding of a specificimmunoglobulin subclass to a specific antigen either foreign of self.Finally, agents can be selected which inhibit the binding of a specificimmunoglobulin subclass to its receptor on specific cell types.

Similarly, agents which affect cytokine production may be selected,generally using two cell systems. For example, cytokine production frommacrophages, monocytes, etc. may be evaluated. Similarly, agents whichmimic cytokines, for example erythropoetin and IL1-17, may be selected,or agents that bind cytokines such as TNF-α, before they bind theirreceptor.

Antigen processing by mononuclear leukocytes (ML) is an important earlystep in the immune system's ability to recognize and eliminate foreignproteins. Candidate agents can be inserted into ML cell lines and agentsselected which alter the intracellular processing of foreign peptidesand sequence of the foreign peptide that is presented to T cells by MLson their cell surface in the context of Class II MHC. One can look formembers of the library that enhance immune responses of a particular Tcell subset (for example, the peptide would in fact work as a vaccine),or look for a library member that binds more tightly to MHC, thusdisplacing naturally occurring peptides, but nonetheless the agent wouldbe less immunogenic (less stimulatory to a specific T cell clone). Thisagent would in fact induce immune tolerance and/or diminish immuneresponses to foreign proteins. This approach could be used intransplantation, autoimmune diseases, and allergic diseases.

The release of inflammatory mediators (cytokines, leukotrienes,prostaglandins, platelet activating factor, histamine, neuropeptides,and other peptide and lipid mediators) is a key element in maintainingand amplifying aberrant immune responses. Candidate libraries can beinserted into MLs, mast cells, eosinophils, and other cellsparticipating in a specific inflammatory response, and bioactivepeptides selected which inhibit the synthesis, release and binding tothe cognate receptor of each of these types of mediators.

In a preferred embodiment, the present methods are useful inbiotechnology applications. Candidate library expression in mammaliancells can also be considered for other pharmaceutical-relatedapplications, such as modification of protein expression, proteinfolding, or protein secretion. One such example would be in commercialproduction of protein pharmaceuticals in CHO or other cells. Candidatelibraries resulting in bioactive peptides which select for an increasedcell growth rate (perhaps peptides mimicking growth factors or acting asagonists of growth factor signal transduction pathways), for pathogenresistance (see previous section), for lack of sialylation orglycosylation (by blocking glycotransferases or rerouting trafficking ofthe protein in the cell), for allowing growth on autoclaved media, orfor growth in serum free media, would all increase productivity anddecrease costs in the production of protein pharmaceuticals.

Random peptides displayed on the surface of circulating cells can beused as tools to identify organ, tissue, and cell specific peptidetargeting sequences. Any cell introduced into the bloodstream of ananimal expressing a library targeted to the cell surface can be selectedfor specific organ and tissue targeting. The bioactive peptide sequenceidentified can then be coupled to an antibody, enzyme, drug, imagingagent or substance for which organ targeting is desired.

Other agents which may be selected using the present inventioninclude: 1) agents which block the activity of transcription factors,using cell lines with reporter genes; 2) agents which block theinteraction of two known proteins in cells, using the absence of normalcellular functions, the mammalian two hybrid system or fluorescenceresonance energy transfer mechanisms for detection; and 3) agents may beidentified by tethering a random peptide to a protein binding region toallow interactions with molecules sterically close, i.e. within asignalling pathway, to localize the effects to a functional area ofinterest.

In a preferred embodiment, the bioactve peptide may also be used in genetherapy. In gene therapy applications, genes encoding the peptide areintroduced into cells in order to achieve in vivo synthesis of atherapeutically effective genetic product. “Gene therapy” includes bothconventional gene therapy where a lasting effect is achieved by a singletreatment, and the administration of gene therapeutic agents, whichinvolves the one time or repeated administration of a therapeuticallyeffective DNA or mRNA.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection [Dzau et al., Trends in Biotechnology 11:205-210 (1993)].In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. U.S.A. 87:3410-3414 (1990). For review of gene marking andgene therapy protocols see Anderson et al., Science 256:808-813 (1992).

Alternatively, an ex vivo approach can be used in which a cell excretinga therapeutically effective peptide may be transplanted into anindividual, for the constant or regulated systemic delivery of thepeptide.

The pharmaceutical compositions of the present invention comprise acompound in a form suitable for administration to a patient. In thepreferred embodiment, the pharmaceutical compositions are in a watersoluble form, such as being present as pharmaceutically acceptablesalts, which is meant to include both acid and base addition salts.“Pharmaceutically acceptable acid addition salt” refers to those saltsthat retain the biological effectiveness of the free bases and that arenot biologically or otherwise undesirable, formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike. “Pharmaceutically acceptable base addition salts” include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium, and magnesium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The compounds can be formulated using pharmaceutically acceptablecarriers into dosages suitable for oral administration. Such carriersenable the compounds of the invention to be formulated as tablets,pills, capsules, liquids, gels, syrups, slurries, and the like for oralingestion.

The administration of the bioactive peptides of the present invention,preferably in the form of a sterile aqueous solution, can be done in avariety of ways, including, but not limited to, orally, subcutaneously,intravenously, intranasally, transdermally, intraperitoneally,intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.In some instances, for example, in the treatment of wounds,inflammation, etc., the peptide may be directly applied as a solution orspray. Depending upon the manner of introduction, the pharmaceuticalcomposition may be formulated in a variety of ways. The concentration ofthe therapeutically active peptide in the formulation may vary fromabout 0.1 to 100 weight %.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference.

EXAMPLES Example 1 Isolation of Inteins with Altered CyclizationActivity

A fluorescent reporter system was designed for quantifying inteincyclization. GFP was split at the loop 3 junction and the translationalorder of the N and C-terminal fragments were reversed (FIG. 12A). Thetermini were held together by a glycine-serine linker. In someconstructs, one-half of the myc epitope was fused onto either side ofthe loop 3 junction (FIG. 12A). The resulting GFP molecules werepositioned with an intein scaffold comprising either wild-type or amutant intein (FIG. 12C).

Mutant intein sequences obtained using PCR mutagenesis were screened foractivity by FACS sorting for increases in fluorescence. Western blotanalysis of several other mutants is shown in FIG. 13. In FIG. 13,several of the mutants had cyclization efficiencies greater thantheparental staring intein, J3.

Example 2 Biasing a Cyclic Peptide to Reduce the Number of Conformers

To test the effects of a fixed proline in a cyclic 7mer, theconformation space of the 7mer cyclic peptide RGDGWS, containing twoflexible glycines was compared with that of cyclic RGPGWS using quencedmolecular dynamics calculations (O'Connor, et al., (1992) J. Med. Chem.,35:2870-81); Mackay, et al., (1989) “The role of energy minimization insimulation stragegies of biomolecular systems”, In Prediction of ProteinStructure and the Principles of Protein Conformation, Fasman, G., ed.,New York, Plemum Press, pp. 317-358).

The lowest 5 kcal energy conformers were collected from a total of atleast 10,000 individual conformers obtained from multiple moleculardynamics trajectories, and compared with each other using the backboneamino acids by overlaying the structures and calculating the root meansquare deviation of these atoms in the best fit overlay using Insightll(Molecular Simulations Inc.).

An example of the cluster graph of the lowest energy conformers for eachpeptide is shown in FIGS. 15 and 16. The root mean square deviation(RMSD, Å) is coded by color, with very similar conformers (RMSD≦Å) inyellow, still highly similar conformers (RMSD between 1-2 Å) in white,similar conformers (RMSD between 2-3 Å) in blue, less similar conformers(RMSD between 3-4 Å) in red, and dissimilar conformers in black (notshown).

For the cyclic peptide SRGDGWS, shown in FIG. 15 (srgdgwsLowest5A.ps),there were 62 low energy conformers. There was one family of verysimilar conformers (yellow square at bottom left) and two families ofquite similar conformers in yellow/white, one roughly in the middle ofthe graph, and one (with only moderately similar conformers) near thetop right corner. These comprised approximately 20 of the 62 conformers.The rest of the low energy conformers were not very similar to eachother, and much of the graph is red or black. Backbone overlaidconformers from most similar family, No. 1, are shown at the lower left.In the lower middle, is family No. 2. these conformers, when overlaidare clearly not similar. Conformers in family No. 3 (lower right), arerather heterogeneous, although not as much as those from the red andblack regions of the graph.

For the cyclic peptide SRGPGWS, representing the substitution of pro forasp 4, the graph of the lowest energy conformers looks quite different(FIG. 16; srgpgwsLowest5B.ps). There is a much larger family of verysimilar conformers (lower left of graph, family No. 1, conformers 1-26).Family No. 2 also has very similar conformers, although they are alldifferent from family No. 1. Even family No. 3, representing over twothirds of all low energy conformers (frames 1-59) contains conformersthat are similar enough to give a blurred donut appearance. Thus,substitution of a single pro for another residue (asp in this case)clearly freezes out two additional families of conformers. As thispeptide has two glycines, the effect of proline on conformationalnarrowing of cyclic peptides with 1 or 0 glycines may be more profound.

1-18. (canceled)
 19. An isolated cell comprising: a cyclic peptideconsisting of four amino acids, wherein at least one of said amino acidsis a Ser, Thr or Cys.
 20. The isolated cell of claim 19, wherein saidcell further comprises: a nucleic acid encoding a fusion proteincomprising, in order: a) a C-terminal intein domain; b) a peptide; andc) a N-terminal intein domain; wherein said fusion protein undergoes areaction to produce said cyclic peptide.
 21. The isolated cell of claim19, wherein said cell is a mammalian cell.
 22. The isolated cell ofclaim 20, wherein said fusion protein is encoded by retroviral vector.23. The isolated cell of claim 20, wherein said cell is a mammaliancell.
 24. An isolated cell comprising a cyclic peptide consisting offour amino acids, wherein at least one of said amino acids is a Ser, Thror Cys, and wherein said cyclic peptide is produced as a result of acyclization reaction of a fusion protein comprising, in order: a) aC-terminal intein domain; b) a peptide; and c) a N-terminal inteindomain.
 25. The isolated cell of claim 24, wherein said cell is amammalian cell.
 26. The isolated cell of claim 24, wherein said fusionprotein is encoded by retroviral vector.